https://proteopedia.org/api.php?action=feedcontributions&feedformat=atom&user=Thomas+GastineauProteopedia - User contributions [en]2026-10-04T08:20:33ZUser contributionsMediaWiki 1.43.8https://proteopedia.org/index.php?title=Cas9&diff=3644196Cas92022-10-17T18:59:42Z<p>Thomas Gastineau: </p>
<hr />
<div>[[Cas9]] is the RNA-guided [[DNA]] [[endonuclease]] used by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated systems to generate double-strand DNA breaks in the invading DNA during an adaptive bacterial immune response.<br />
<br />
See also [[Cas9 (hebrew)]].<br />
<br />
The CRISPR-associated endonuclease [[Cas9]] has been exploited for use in genome editing systems. In such systems, an engineered single-guide RNA (sgRNA) is used to target double-stranded breaks in genomic DNA. Depending on what repair pathway is triggered, often dictated by the inclusion of additional engineered components, the targeted site either is disrupted or incorporates additional genetic sequences. <br />
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[[Image:Layout for schematic and structure with structure.png|660px]]<br />
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<center><html5media height=“315” width=“560” frameborder="0" allowfullscreen>https://www.youtube.com/embed/TdBAHexVYzc</html5media></center><br />
<center>Geneticist and 2020 Nobel laureate [http://rna.berkeley.edu/ Jennifer Doudna], from UC Berkeley, is one of the co-inventors of the groundbreaking </center><br />
<center>new technology for editing genes, called CRISPR-Cas9. The tool allows scientists</center><br />
<center>to make precise edits to DNA strands, which could lead to treatments for genetic diseases … </center><br />
<center>but could also be used to create so-called "designer babies." </center><br />
<center>Doudna reviews how CRISPR-Cas9 works — and asks the scientific community </center><br />
<center>to pause and discuss the ethics of this new tool.</center><br />
<br />
<center><br>Microbiologist and 2020 Nobel laureate [https://www.emmanuelle-charpentier-lab.org/ Emanuelle Charpentier], from Max Planck Institute for Infection Biology in Berlin, is one of the co-inventors of the groundbreaking </center><br />
<center>new technology for editing genes, called CRISPR-Cas9. The tool allows scientists</center><br />
<center>to make precise edits to DNA strands, which could lead to treatments for genetic diseases … </center><br />
<center>but could also be used to create so-called "designer babies." </center><br />
<center>Charpentier reviews how CRISPR-Cas9 works in [https://www.cnn.com/videos/tech/2016/04/27/crispr-cas9-explainer-natpkg.cnn/video/playlists/fertility-health/ this 2016 talk].<br></center><br />
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<center><html5media height=“315” width=“560” frameborder="0" allowfullscreen>https://www.youtube.com/embed/MnYppmstxIs</html5media><br />
<br>This video, by Paul Andersen, explains how the CRISPR/Cas immune system <br>was identified in bacteria and how the CRISPR/Cas9 system was developed to edit genomes.</center><br />
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<center><html5media height=“315” width=“560” frameborder="0" allowfullscreen>https://www.youtube.com/embed/2pp17E4E-O8</html5media></center><br />
<center>Movie from the [http://mcgovern.mit.edu/ McGovern Institute for Brain Research at MIT]</center><br />
<center>This animation depicts the CRISPR-Cas9 method for genome editing: <br>a powerful new technology with many applications in biomedical research, <br>including the potential to treat human genetic disease.</center><br />
<br />
Articles in Proteopedia concerning Cas9 include:<br />
* [[Cas9 Sandbox|Brett Thumm's Student Project page on Cas9]]<br />
<br />
==3D structures of Cas9==<br />
<br />
[[Image:4un3 labeled.png|right|390px]]<br />
<br />
===''Streptococcus pyogenes'' Cas9===<br />
<br />
* [[4un3]], [[4un4]], and [[4un5]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br />
* [[4oo8]] - ''S. pyogenes'' Cas9 bound to sgRNA and target DNA<br />
* [[4cmp]]<br />
* [[4cmq]] - Mn<sup>2+</sup>-bound ''S. pyogenes'' Cas9<br />
<br />
=== ''Actinomyces naeslundii'' Cas9=== <br />
*[[4oge]]<br />
*[[4ogc]] - Mn<sup>2+</sup>-bound ''A.s naeslundii'' Cas9<br />
<br />
==See Also==<br />
[[H-N-H motif]] <br/><br />
<br />
<br />
== References ==<br />
<references/><br />
<br />
[[Category: Crispr]]<br />
[[Category: Crispr-associated]]<br />
[[Category: endonuclease]]<br />
<br />
==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections. CRISPR is a form of acquired immunity used by bacteria. CRISPR stands for clustered regularly interspaced short palindromic repeats because the bacterial genome includes genetic sequences clustered together from bacteriophages of previous infections that are used by Cas9 to cut viral DNA. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA, rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single-stranded guide RNA (sgRNA) to complimentarily bind the target DNA that will create a double stranded DNA cut in the proper location. The target DNA must also have a PAM sequence to bind for Cas9 to cut target DNA. The PAM sequence stands for protospacer adjacent motif and is downstream from the cut site of the nuclease. The PAM sequence acts as a two-factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. <br />
<br />
The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the <scene name='92/925538/Lobes_and_linkers/16'>REC lobe</scene> (residues 41–425) and <scene name='92/925538/Lobes_and_linkers/15'>NUC lobe</scene> (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WED, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The NUC lobe stands for the nuclease lobe and each of these domains are a part of the protein that allow Cas9 to cuts the target DNA <ref>PMID:15596446</ref><ref>PMID:24634220</ref><ref>PMID:24529477</ref>. The REC lobe stands for the recognition lobe is responsible for recognizing the target DNA present causing a conformational change in the HNH domain locking the HNH domain into the cleavage site <ref>PMID:30555184</ref>. Cas9 has four main mechanisms that are important for successful cleavage, including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting the heteroduplex into the central channel between the REC and NUC lobes. A heteroduplex is the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe and bridge helix interacts with the seed region of the <scene name='92/925538/Lobes_and_linkers/17'>sgRNA</scene> (C13-C20). The positive charged residues on the bridge helix (Asn44, Arg48, Arg51, Arg55, Arg59, and Arg60) and REC lobe (Arg116, Arg165, Asn169, and Arg209) interact with the negative phosphate backbone. The seed region is in the <scene name='92/925538/Lobes_and_linkers/8'>A-form conformation</scene>, so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the <scene name='92/925538/Lobes_and_linkers/21'>sugar-phosphate backbone</scene> (the hydrogen bonds are shown as black dashes). The target DNA binds to the <scene name='92/925538/Lobes_and_linkers/9'>REC lobe and RuvC domain</scene> for the proper conformation for base paring between the target DNA and sgRNA<ref name="Cas9" />.<br />
== Recognition of the PAM sequence ==<br />
For the recognition of the <scene name='92/925538/Lobes_and_linkers/22'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through hydrogen bonds as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. This PAM sequence is differnt that other PAM sequences like the one found in SpCas9 (5'-NGG-3'). The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.<br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the <scene name='92/925538/Lobes_and_linkers/20'>REC lobe and WED domain</scene>. The WED contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat: anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="Cas9" />.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, <scene name='92/925538/Lobes_and_linkers/12'>RuvC and HNH</scene> are involved in endonuclease activity. RuvC uses two manganese ions to cleave the non-target DNA through manganese coordinating with the phosphate backbone and aspartic acid residues. These phosphate oxygens coordinated with the manganese makes the phosphate a greater target for nucleophillic attack.A histidine then acts as a base to create a hydroxide nucleophile that attacks the phosphate bond and cleaves the non-target DNA. The binding of the RuvC to the target DNA changes the conformation of a linker protein region between the RuvC domain and the HNH domain. The conformational change of the linker brings the HNH domain close enough to the target DNA to cut the DNA. This linker conformational change is not present in the crystal structure, therefore the HNH appears to be far from the target DNA and in an inactive state. The HNH follows a similar mechanism as to RuvC using a histidine base to create a hydroxide ion nucleophile that attacks the phosphate bond using one manganese ion instead of two. This is modeled as manganese however, it magnesium is used in cells <ref name="Cas9" />.<br />
</structuresection><br />
==References==<br />
<br />
<references /></div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3644195Sandbox Reserved 17502022-10-17T18:58:37Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections. CRISPR is a form of acquired immunity used by bacteria. CRISPR stands for clustered regularly interspaced short palindromic repeats because the bacterial genome includes genetic sequences clustered together from bacteriophages of previous infections that are used by Cas9 to cut viral DNA. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA, rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single-stranded guide RNA (sgRNA) to complimentarily bind the target DNA that will create a double stranded DNA cut in the proper location. The target DNA must also have a PAM sequence to bind for Cas9 to cut target DNA. The PAM sequence stands for protospacer adjacent motif and is downstream from the cut site of the nuclease. The PAM sequence acts as a two-factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. <br />
<br />
The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the <scene name='92/925538/Lobes_and_linkers/16'>REC lobe</scene> (residues 41–425) and <scene name='92/925538/Lobes_and_linkers/15'>NUC lobe</scene> (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WED, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The NUC lobe stands for the nuclease lobe and each of these domains are a part of the protein that allow Cas9 to cuts the target DNA <ref>PMID:15596446</ref><ref>PMID:24634220</ref><ref>PMID:24529477</ref>. The REC lobe stands for the recognition lobe is responsible for recognizing the target DNA present causing a conformational change in the HNH domain locking the HNH domain into the cleavage site <ref>PMID:30555184</ref>. Cas9 has four main mechanisms that are important for successful cleavage, including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting the heteroduplex into the central channel between the REC and NUC lobes. A heteroduplex is the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe and bridge helix interacts with the seed region of the <scene name='92/925538/Lobes_and_linkers/17'>sgRNA</scene> (C13-C20). The positive charged residues on the bridge helix (Asn44, Arg48, Arg51, Arg55, Arg59, and Arg60) and REC lobe (Arg116, Arg165, Asn169, and Arg209) interact with the negative phosphate backbone. The seed region is in the <scene name='92/925538/Lobes_and_linkers/8'>A-form conformation</scene>, so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the <scene name='92/925538/Lobes_and_linkers/21'>sugar-phosphate backbone</scene> (the hydrogen bonds are shown as black dashes). The target DNA binds to the <scene name='92/925538/Lobes_and_linkers/9'>REC lobe and RuvC domain</scene> for the proper conformation for base paring between the target DNA and sgRNA<ref name="Cas9" />.<br />
== Recognition of the PAM sequence ==<br />
For the recognition of the <scene name='92/925538/Lobes_and_linkers/22'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through hydrogen bonds as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. This PAM sequence is differnt that other PAM sequences like the one found in SpCas9 (5'-NGG-3'). The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.<br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the <scene name='92/925538/Lobes_and_linkers/20'>REC lobe and WED domain</scene>. The WED contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat: anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="Cas9" />.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, <scene name='92/925538/Lobes_and_linkers/12'>RuvC and HNH</scene> are involved in endonuclease activity. RuvC uses two manganese ions to cleave the non-target DNA through manganese coordinating with the phosphate backbone and aspartic acid residues. These phosphate oxygens coordinated with the manganese makes the phosphate a greater target for nucleophillic attack.A histidine then acts as a base to create a hydroxide nucleophile that attacks the phosphate bond and cleaves the non-target DNA. The binding of the RuvC to the target DNA changes the conformation of a linker protein region between the RuvC domain and the HNH domain. The conformational change of the linker brings the HNH domain close enough to the target DNA to cut the DNA. This linker conformational change is not present in the crystal structure, therefore the HNH appears to be far from the target DNA and in an inactive state. The HNH follows a similar mechanism as to RuvC using a histidine base to create a hydroxide ion nucleophile that attacks the phosphate bond using one manganese ion instead of two. This is modeled as manganese however, it magnesium is used in cells <ref name="Cas9" />.<br />
</structuresection><br />
==References==<br />
<br />
<references /></div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3644194Sandbox Reserved 17502022-10-17T18:56:34Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections. CRISPR is a form of acquired immunity used by bacteria. CRISPR stands for clustered regularly interspaced short palindromic repeats because the bacterial genome includes genetic sequences clustered together from bacteriophages of previous infections that are used by Cas9 to cut viral DNA. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA, rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single-stranded guide RNA (sgRNA) to complimentarily bind the target DNA that will create a double stranded DNA cut in the proper location. The target DNA must also have a PAM sequence to bind for Cas9 to cut target DNA. The PAM sequence stands for protospacer adjacent motif and is downstream from the cut site of the nuclease. The PAM sequence acts as a two-factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. <br />
<br />
The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the <scene name='92/925538/Lobes_and_linkers/16'>REC lobe</scene> (residues 41–425) and <scene name='92/925538/Lobes_and_linkers/15'>NUC lobe</scene> (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WED, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The NUC lobe stands for the nuclease lobe and each of these domains are a part of the protein that allow Cas9 to cuts the target DNA <ref>PMID:15596446</ref><ref>PMID:24634220</ref><ref>PMID:24529477</ref>. The REC lobe stands for the recognition lobe is responsible for recognizing the target DNA present causing a conformational change in the HNH domain locking the HNH domain into the cleavage site <ref>PMID:30555184</ref>. Cas9 has four main mechanisms that are important for successful cleavage, including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe and bridge helix interacts with the seed region of the <scene name='92/925538/Lobes_and_linkers/17'>sgRNA</scene> (C13-C20). The positive charged residues on the bridge helix (Asn44, Arg48, Arg51, Arg55, Arg59, and Arg60) and REC lobe (Arg116, Arg165, Asn169, and Arg209) interact with the negative phosphate backbone. The seed region is in the <scene name='92/925538/Lobes_and_linkers/8'>A-form conformation</scene>, so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the <scene name='92/925538/Lobes_and_linkers/21'>sugar-phosphate backbone</scene> (the hydrogen bonds are shown as black dashes). The target DNA binds to the <scene name='92/925538/Lobes_and_linkers/9'>REC lobe and RuvC domain</scene> for the proper conformation for base paring between the target DNA and sgRNA<ref name="Cas9" />.<br />
== Recognition of the PAM sequence ==<br />
For the recognition of the <scene name='92/925538/Lobes_and_linkers/22'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through hydrogen bonds as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. This PAM sequence is differnt that other PAM sequences like the one found in SpCas9 (5'-NGG-3'). The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.<br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the <scene name='92/925538/Lobes_and_linkers/20'>REC lobe and WED domain</scene>. The WED contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat: anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="Cas9" />.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, <scene name='92/925538/Lobes_and_linkers/12'>RuvC and HNH</scene> are involved in endonuclease activity. RuvC uses two manganese ions to cleave the non-target DNA through manganese coordinating with the phosphate backbone and aspartic acid residues. These phosphate oxygens coordinated with the manganese makes the phosphate a greater target for nucleophillic attack.A histidine then acts as a base to create a hydroxide nucleophile that attacks the phosphate bond and cleaves the non-target DNA. The binding of the RuvC to the target DNA changes the conformation of a linker protein region between the RuvC domain and the HNH domain. The conformational change of the linker brings the HNH domain close enough to the target DNA to cut the DNA. This linker conformational change is not present in the crystal structure, therefore the HNH appears to be far from the target DNA and in an inactive state. The HNH follows a similar mechanism as to RuvC using a histidine base to create a hydroxide ion nucleophile that attacks the phosphate bond using one manganese ion instead of two. This is modeled as manganese however, it magnesium is used in cells <ref name="Cas9" />.<br />
</structuresection><br />
==References==<br />
<br />
<references /></div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3644193Sandbox Reserved 17502022-10-17T18:53:22Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections. CRISPR is a form of acquired immunity used by bacteria. CRISPR stands for clustered regularly interspaced short palindromic repeats because the bacterial genome includes genetic sequences clustered together from bacteriophages of previous infections that are used by Cas9 to cut viral DNA. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA, rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single-stranded guide RNA (sgRNA) to complimentarily bind the target DNA that will create a double stranded DNA cut in the proper location. The target DNA must also have a PAM sequence to bind for Cas9 to cut target DNA. The PAM sequence stands for protospacer adjacent motif and is downstream from the cut site of the nuclease. The PAM sequence acts as a two-factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. <br />
<br />
The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the <scene name='92/925538/Lobes_and_linkers/16'>REC lobe</scene> (residues 41–425) and <scene name='92/925538/Lobes_and_linkers/15'>NUC lobe</scene> (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WED, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The NUC lobe stands for the nuclease lobe and each of these domains are a part of the protein that cuts the target DNA <ref>PMID:15596446</ref><ref>PMID:24634220</ref><ref>PMID:24529477</ref>. The REC lobe stands for the recognition lobe is responsible for recognizing the target DNA present causing a conformational change in the HNH domain locking the HNH domain into the cleavage site <ref>PMID:30555184</ref>. Cas9 has four main mechanisms that are important for successful cleavage, including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe and bridge helix interacts with the seed region of the <scene name='92/925538/Lobes_and_linkers/17'>sgRNA</scene> (C13-C20). The positive charged residues on the bridge helix (Asn44, Arg48, Arg51, Arg55, Arg59, and Arg60) and REC lobe (Arg116, Arg165, Asn169, and Arg209) interact with the negative phosphate backbone. The seed region is in the <scene name='92/925538/Lobes_and_linkers/8'>A-form conformation</scene>, so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the <scene name='92/925538/Lobes_and_linkers/21'>sugar-phosphate backbone</scene> (the hydrogen bonds are shown as black dashes). The target DNA binds to the <scene name='92/925538/Lobes_and_linkers/9'>REC lobe and RuvC domain</scene> for the proper conformation for base paring between the target DNA and sgRNA<ref name="Cas9" />.<br />
== Recognition of the PAM sequence ==<br />
For the recognition of the <scene name='92/925538/Lobes_and_linkers/22'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through hydrogen bonds as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. This PAM sequence is differnt that other PAM sequences like the one found in SpCas9 (5'-NGG-3'). The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.<br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the <scene name='92/925538/Lobes_and_linkers/20'>REC lobe and WED domain</scene>. The WED contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat: anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="Cas9" />.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, <scene name='92/925538/Lobes_and_linkers/12'>RuvC and HNH</scene> are involved in endonuclease activity. RuvC uses two manganese ions to cleave the non-target DNA through manganese coordinating with the phosphate backbone and aspartic acid residues. These phosphate oxygens coordinated with the manganese makes the phosphate a greater target for nucleophillic attack.A histidine then acts as a base to create a hydroxide nucleophile that attacks the phosphate bond and cleaves the non-target DNA. The binding of the RuvC to the target DNA changes the conformation of a linker protein region between the RuvC domain and the HNH domain. The conformational change of the linker brings the HNH domain close enough to the target DNA to cut the DNA. This linker confromational change is not present in the crystal structure, therefore the HNH appears to be far from the target DNA. The HNH follows a similar mechanism as to RuvC using a histidine base to create a hydroxide ion nucleophile that attacks the phosphate bond using one manganese ion instead of two. This is modeled as manganese however, it magnesium is used in cells <ref name="Cas9" />.<br />
</structuresection><br />
==References==<br />
<br />
<references /></div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3644192Sandbox Reserved 17502022-10-17T18:51:28Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections. CRISPR is a form of acquired immunity used by bacteria. CRISPR stands for clustered regularly interspaced short palindromic repeats because the bacterial genome includes genetic sequences clustered together from bacteriophages of previous infections that are used as by Cas9 to cut viral DNA. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA, rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single-stranded guide RNA (sgRNA) to complimentarily bind the target DNA that will create a double stranded DNA cut in the proper location. The target DNA must also have a PAM sequence to bind for Cas9 to cut target DNA. The PAM sequence stands for protospacer adjacent motif and is downstream from the cut site of the nuclease. The PAM sequence acts as a two-factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. <br />
<br />
The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the <scene name='92/925538/Lobes_and_linkers/16'>REC lobe</scene> (residues 41–425) and <scene name='92/925538/Lobes_and_linkers/15'>NUC lobe</scene> (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WED, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The NUC lobe stands for the nuclease lobe and each of these domains are a part of the protein that cuts the target DNA <ref>PMID:15596446</ref><ref>PMID:24634220</ref><ref>PMID:24529477</ref>. The REC lobe stands for the recognition lobe is responsible for recognizing the target DNA present causing a conformational change in the HNH domain locking the HNH domain into the cleavage site <ref>PMID:30555184</ref>. Cas9 has four main mechanisms that are important for successful cleavage, including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe and bridge helix interacts with the seed region of the <scene name='92/925538/Lobes_and_linkers/17'>sgRNA</scene> (C13-C20). The positive charged residues on the bridge helix (Asn44, Arg48, Arg51, Arg55, Arg59, and Arg60) and REC lobe (Arg116, Arg165, Asn169, and Arg209) interact with the negative phosphate backbone. The seed region is in the <scene name='92/925538/Lobes_and_linkers/8'>A-form conformation</scene>, so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the <scene name='92/925538/Lobes_and_linkers/21'>sugar-phosphate backbone</scene> (the hydrogen bonds are shown as black dashes). The target DNA binds to the <scene name='92/925538/Lobes_and_linkers/9'>REC lobe and RuvC domain</scene> for the proper conformation for base paring between the target DNA and sgRNA<ref name="Cas9" />.<br />
== Recognition of the PAM sequence ==<br />
For the recognition of the <scene name='92/925538/Lobes_and_linkers/22'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through hydrogen bonds as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. This PAM sequence is differnt that other PAM sequences like the one found in SpCas9 (5'-NGG-3'). The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.<br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the <scene name='92/925538/Lobes_and_linkers/20'>REC lobe and WED domain</scene>. The WED contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat: anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="Cas9" />.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, <scene name='92/925538/Lobes_and_linkers/12'>RuvC and HNH</scene> are involved in endonuclease activity. RuvC uses two manganese ions to cleave the non-target DNA through manganese coordinating with the phosphate backbone and aspartic acid residues. These phosphate oxygens coordinated with the manganese makes the phosphate a greater target for nucleophillic attack.A histidine then acts as a base to create a hydroxide nucleophile that attacks the phosphate bond and cleaves the non-target DNA. The binding of the RuvC to the target DNA changes the conformation of a linker protein region between the RuvC domain and the HNH domain. The conformational change of the linker brings the HNH domain close enough to the target DNA to cut the DNA. This linker confromational change is not present in the crystal structure, therefore the HNH appears to be far from the target DNA. The HNH follows a similar mechanism as to RuvC using a histidine base to create a hydroxide ion nucleophile that attacks the phosphate bond using one manganese ion instead of two. This is modeled as manganese however, it magnesium is used in cells <ref name="Cas9" />.<br />
</structuresection><br />
==References==<br />
<br />
<references /></div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3644191Sandbox Reserved 17502022-10-17T18:37:04Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections. CRISPR is a form of acquired immunity used by bacteria. CRISPR stands for clustered regularly interspaced short palindromic repeats because the bacterial genome includes genetic sequences clustered together from bacteriophages of previous infections that are used as by Cas9 to cut viral DNA. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA, rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single-stranded guide RNA (sgRNA) to complimentarily bind the target DNA that will create a double stranded DNA cut in the proper location. The target DNA must also have a PAM sequence to bind for Cas9 to cut target DNA. The PAM sequence stands for protospacer adjacent motif and is downstream from the cut site of the nuclease. The PAM sequence acts as a two-factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. <br />
<br />
The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the <scene name='92/925538/Lobes_and_linkers/16'>REC lobe</scene> (residues 41–425) and <scene name='92/925538/Lobes_and_linkers/15'>NUC lobe</scene> (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WED, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The NUC lobe stands for the nuclease lobe and each of these domains are a part of the protein that cuts the target DNA <ref>PMID:15596446</ref><ref>PMID:24634220</ref><ref>PMID:24529477</ref>. The REC lobe stands for the recognition lobe is responsible for recognizing the target DNA present causing a conformational change in the HNH domain locking the HNH domain into the cleavage site <ref>PMID:30555184</ref>. Cas9 has four main mechanisms that are important for successful cleavage, including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe and bridge helix interacts with the seed region of the <scene name='92/925538/Lobes_and_linkers/17'>sgRNA</scene> (C13-C20). The positive charged residues on the bridge helix (Asn44, Arg48, Arg51, Arg55, Arg59, and Arg60) and REC lobe (Arg116, Arg165, Asn169, and Arg209) interact with the negative phosphate backbone. The seed region is in the <scene name='92/925538/Lobes_and_linkers/8'>A-form conformation</scene>, so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the <scene name='92/925538/Lobes_and_linkers/21'>sugar-phosphate backbone</scene> (the hydrogen bonds are shown as black dashes). The target DNA binds to the <scene name='92/925538/Lobes_and_linkers/9'>REC lobe and RuvC domain</scene> for the proper conformation for base paring between the target DNA and sgRNA<ref name="Cas9" />.<br />
== Recognition of the PAM sequence ==<br />
For the recognition of the <scene name='92/925538/Lobes_and_linkers/22'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through hydrogen bonds as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. This PAM sequence is differnt that other PAM sequences like the one found in SpCas9 (5'-NGG-3'). The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.<br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the <scene name='92/925538/Lobes_and_linkers/20'>REC lobe and WED domain</scene>. The WED contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat: anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="Cas9" />.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, <scene name='92/925538/Lobes_and_linkers/12'>RuvC and HNH</scene> are involved in endonuclease activity. RuvC uses two manganese ions to cleave the non-target DNA through manganese coordinating with the phosphate backbone and aspartic acid residues. These phosphate oxygens coordinated with the manganese makes the phosphate a greater target for nucleophillic attack.A histidine then acts as a base to create a hydroxide nucleophile that attacks the phosphate bond and cleaves the non-target DNA. The binding of the RuvC to the target DNA changes the conformation of a linker protein region between the RuvC domain and the HNH domain. The conformational change of the linker brings the HNH domain close enough to the target DNA to cut the DNA. The HNH follows a similar mechanism as to RuvC using a histidine base to create a hydroxide ion nucleophile that attacks the phosphate bond using one manganese ion instead of two. This is modeled as manganese however, it magnesium is used in cells <ref name="Cas9" />.<br />
</structuresection><br />
==References==<br />
<br />
<references /></div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3644190Sandbox Reserved 17502022-10-17T18:32:21Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections. CRISPR is a form of acquired immunity used by bacteria. CRISPR stands for clustered regularly interspaced short palindromic repeats because the bacterial genome includes genetic sequences clustered together from bacteriophages of previous infections that are used as by Cas9 to cut viral DNA. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA, rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single-stranded guide RNA (sgRNA) to complimentarily bind the target DNA that will create a double stranded DNA cut in the proper location. The target DNA must also have a PAM sequence to bind for Cas9 to cut target DNA. The PAM sequence stands for protospacer adjacent motif and is downstream from the cut site of the nuclease. The PAM sequence acts as a two-factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. <br />
<br />
The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the <scene name='92/925538/Lobes_and_linkers/16'>REC lobe</scene> (residues 41–425) and <scene name='92/925538/Lobes_and_linkers/15'>NUC lobe</scene> (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WED, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The NUC lobe stands for the nuclease lobe and each of these domains are a part of the protein that cuts the target DNA <ref>PMID:15596446</ref><ref>PMID:24634220</ref><ref>PMID:24529477</ref>. The REC lobe stands for the recognition lobe is responsible for recognizing the target DNA present causing a conformational change in the HNH domain locking the HNH domain into the cleavage site <ref>PMID:30555184</ref>. Cas9 has four main mechanisms that are important for successful cleavage, including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe and bridge helix interacts with the seed region of the <scene name='92/925538/Lobes_and_linkers/17'>sgRNA</scene> (C13-C20). The positive charged residues on the bridge helix (Asn44, Arg48, Arg51, Arg55, Arg59, and Arg60) and REC lobe (Arg116, Arg165, Asn169, and Arg209) interact with the negative phosphate backbone. The seed region is in the <scene name='92/925538/Lobes_and_linkers/8'>A-form conformation</scene>, so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the <scene name='92/925538/Lobes_and_linkers/21'>sugar-phosphate backbone</scene> (the hydrogen bonds are shown as black dashes). The target DNA binds to the <scene name='92/925538/Lobes_and_linkers/9'>REC lobe and RuvC domain</scene> for the proper conformation for base paring between the target DNA and sgRNA<ref name="Cas9" />.<br />
== Recognition of the PAM sequence ==<br />
For the recognition of the <scene name='92/925538/Lobes_and_linkers/19'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through hydrogen bonds as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. This PAM sequence is differnt that other PAM sequences like the one found in SpCas9 (5'-NGG-3'). The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.<br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the <scene name='92/925538/Lobes_and_linkers/20'>REC lobe and WED domain</scene>. The WED contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat: anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="Cas9" />.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, <scene name='92/925538/Lobes_and_linkers/12'>RuvC and HNH</scene> are involved in endonuclease activity. RuvC uses two manganese ions to cleave the non-target DNA through manganese coordinating with the phosphate backbone and aspartic acid residues. These phosphate oxygens coordinated with the manganese makes the phosphate a greater target for nucleophillic attack.A histidine then acts as a base to create a hydroxide nucleophile that attacks the phosphate bond and cleaves the non-target DNA. The binding of the RuvC to the target DNA changes the conformation of a linker protein region between the RuvC domain and the HNH domain. The conformational change of the linker brings the HNH domain close enough to the target DNA to cut the DNA. The HNH follows a similar mechanism as to RuvC using a histidine base to create a hydroxide ion nucleophile that attacks the phosphate bond using one manganese ion instead of two. This is modeled as manganese however, it magnesium is used in cells <ref name="Cas9" />.<br />
</structuresection><br />
==References==<br />
<br />
<references /></div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3644189Sandbox Reserved 17502022-10-17T18:16:37Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections. CRISPR is a form of acquired immunity used by bacteria. CRISPR stands for clustered regularly interspaced short palindromic repeats because the bacterial genome includes genetic sequences clustered together from bacteriophages of previous infections that are used as by Cas9 to cut viral DNA. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA, rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single-stranded guide RNA (sgRNA) to complimentarily bind the target DNA that will create a double stranded DNA cut in the proper location. The target DNA must also have a PAM sequence to bind for Cas9 to cut target DNA. The PAM sequence stands for protospacer adjacent motif and is downstream from the cut site of the nuclease. The PAM sequence acts as a two-factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. <br />
<br />
The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the <scene name='92/925538/Lobes_and_linkers/16'>REC lobe</scene> (residues 41–425) and <scene name='92/925538/Lobes_and_linkers/15'>NUC lobe</scene> (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WED, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The NUC lobe stands for the nuclease lobe and each of these domains are a part of the protein that cuts the target DNA <ref>PMID:15596446</ref><ref>PMID:24634220</ref><ref>PMID:24529477</ref>. The REC lobe stands for the recognition lobe is responsible for recognizing the target DNA present causing a conformational change in the HNH domain locking the HNH domain into the cleavage site <ref>PMID:30555184</ref>. Cas9 has four main mechanisms that are important for successful cleavage, including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe and bridge helix interacts with the seed region of the <scene name='92/925538/Lobes_and_linkers/17'>sgRNA</scene> (C13-C20). The positive charged residues on the bridge helix (Asn44, Arg48, Arg51, Arg55, Arg59, and Arg60) and REC lobe (Arg116, Arg165, Asn169, and Arg209) interact with the negative phosphate backbone. The seed region is in the <scene name='92/925538/Lobes_and_linkers/8'>A-form conformation</scene>, so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the <scene name='92/925538/Lobes_and_linkers/18'>phosphate backbone</scene>. The target DNA binds to the <scene name='92/925538/Lobes_and_linkers/9'>REC lobe and RuvC domain</scene> for the proper conformation for base paring between the target DNA and sgRNA<ref name="Cas9" />.<br />
== Recognition of the PAM sequence ==<br />
For the recognition of the <scene name='92/925538/Lobes_and_linkers/19'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through hydrogen bonds as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. This PAM sequence is differnt that other PAM sequences like the one found in SpCas9 (5'-NGG-3'). The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.<br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the <scene name='92/925538/Lobes_and_linkers/20'>REC lobe and WED domain</scene>. The WED contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat: anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="Cas9" />.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, <scene name='92/925538/Lobes_and_linkers/12'>RuvC and HNH</scene> are involved in endonuclease activity. RuvC uses two manganese ions to cleave the non-target DNA through manganese coordinating with the phosphate backbone and aspartic acid residues. These phosphate oxygens coordinated with the manganese makes the phosphate a greater target for nucleophillic attack.A histidine then acts as a base to create a hydroxide nucleophile that attacks the phosphate bond and cleaves the non-target DNA. The binding of the RuvC to the target DNA changes the conformation of a linker protein region between the RuvC domain and the HNH domain. The conformational change of the linker brings the HNH domain close enough to the target DNA to cut the DNA. The HNH follows a similar mechanism as to RuvC using a histidine base to create a hydroxide ion nucleophile that attacks the phosphate bond using one manganese ion instead of two. This is modeled as manganese however, it magnesium is used in cells <ref name="Cas9" />.<br />
</structuresection><br />
==References==<br />
<br />
<references /></div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3644188Sandbox Reserved 17502022-10-17T16:44:32Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections. CRISPR is a form of acquired immunity used by bacteria. CRISPR stands for clustered regularly interspaced short palindromic repeats because the bacterial genome includes genetic sequences clustered together from bacteriophages of previous infections that are used as by Cas9 to cut viral DNA. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA, rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single-stranded guide RNA (sgRNA) to complimentarily bind the target DNA that will create a double stranded DNA cut in the proper location. The target DNA must also have a PAM sequence to bind for Cas9 to cut target DNA. The PAM sequence stands for protospacer adjacent motif and is downstream from the cut site of the nuclease. The PAM sequence acts as a two-factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. <br />
<br />
The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the <scene name='92/925538/Lobes_and_linkers/16'>REC lobe</scene> (residues 41–425) and <scene name='92/925538/Lobes_and_linkers/15'>NUC lobe</scene> (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WED, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The NUC lobe stands for the nuclease lobe and each of these domains are a part of the protein that cuts the target DNA <ref>PMID:15596446</ref><ref>PMID:24634220</ref><ref>PMID:24529477</ref>. The REC lobe stands for the recognition lobe is responsible for recognizing the target DNA present causing a conformational change in the HNH domain locking the HNH domain into the cleavage site <ref>PMID:30555184</ref>. Cas9 has four main mechanisms that are important for successful cleavage, including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe and bridge helix interacts with the seed region of the <scene name='92/925538/Lobes_and_linkers/17'>sgRNA</scene> (C13-C20). The seed region is in the <scene name='92/925538/Lobes_and_linkers/8'>A-form conformation</scene>, so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the <scene name='92/925538/Lobes_and_linkers/18'>phosphate backbone</scene>. The target DNA binds to the <scene name='92/925538/Lobes_and_linkers/9'>REC lobe and RuvC domain</scene> for the proper conformation for base paring between the target DNA and sgRNA<ref name="Cas9" />.<br />
== Recognition of the PAM sequence ==<br />
For the recognition of the <scene name='92/925538/Lobes_and_linkers/19'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through hydrogen bonds as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. This PAM sequence is differnt that other PAM sequences like the one found in SpCas9 (5'-NGG-3'). The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.<br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the <scene name='92/925538/Lobes_and_linkers/20'>REC lobe and WED domain</scene>. The WED contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat: anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="Cas9" />.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, <scene name='92/925538/Lobes_and_linkers/12'>RuvC and HNH</scene> are involved in endonuclease activity. RuvC uses two manganese ions to cleave the non-target DNA through manganese coordinating with the phosphate backbone and aspartic acid residues. These phosphate oxygens coordinated with the manganese makes the phosphate a greater target for nucleophillic attack.A histidine then acts as a base to create a hydroxide nucleophile that attacks the phosphate bond and cleaves the non-target DNA. The binding of the RuvC to the target DNA changes the conformation of a linker protein region between the RuvC domain and the HNH domain. The conformational change of the linker brings the HNH domain close enough to the target DNA to cut the DNA. The HNH follows a similar mechanism as to RuvC using a histidine base to create a hydroxide ion nucleophile that attacks the phosphate bond using one manganese ion instead of two. This is modeled as manganese however, it magnesium is used in cells <ref name="Cas9" />.<br />
</structuresection><br />
==References==<br />
<br />
<references /></div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3644187Sandbox Reserved 17502022-10-17T16:40:31Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections. CRISPR is a form of acquired immunity used by bacteria. CRISPR stands for clustered regularly interspaced short palindromic repeats because the bacterial genome includes genetic sequences clustered together from bacteriophages of previous infections that are used as by Cas9 to cut viral DNA. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA, rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single-stranded guide RNA (sgRNA) to complimentarily bind the target DNA that will create a double stranded DNA cut in the proper location. The target DNA must also have a PAM sequence to bind for Cas9 to cut target DNA. The PAM sequence stands for protospacer adjacent motif and is downstream from the cut site of the nuclease. The PAM sequence acts as a two-factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. <br />
<br />
The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the <scene name='92/925538/Lobes_and_linkers/16'>REC lobe</scene> (residues 41–425) and <scene name='92/925538/Lobes_and_linkers/15'>NUC lobe</scene> (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WED, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The NUC lobe stands for the nuclease lobe and each of these domains are a part of the protein that cuts the target DNA <ref>PMID:15596446</ref><ref>PMID:24634220</ref><ref>PMID:24529477</ref>. The REC lobe stands for the recognition lobe is responsible for recognizing the target DNA present causing a conformational change in the HNH domain locking the HNH domain into the cleavage site <ref>PMID:30555184</ref>. Cas9 has four main mechanisms that are important for successful cleavage, including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe and bridge helix interacts with the seed region of the <scene name='92/925538/Lobes_and_linkers/17'>sgRNA</scene> (C13-C20). The seed region is in the <scene name='92/925538/Lobes_and_linkers/8'>A-form conformation</scene>, so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the <scene name='92/925538/Lobes_and_linkers/18'>phosphate backbone</scene>. The target DNA binds to the <scene name='92/925538/Lobes_and_linkers/9'>REC lobe and RuvC domain</scene> for the proper conformation for base paring between the target DNA and sgRNA<ref name="Cas9" />.<br />
== Recognition of the PAM sequence ==<br />
For the recognition of the <scene name='92/925538/Lobes_and_linkers/19'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through hydrogen bonds as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. This PAM sequence is differnt that other PAM sequences like the one found in SpCas9 (5'-NGG-3'). The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.<br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the <scene name='92/925538/Lobes_and_linkers/20'>REC lobe and WED domain</scene>. The WED contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat: anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="Cas9" />.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, <scene name='92/925538/Lobes_and_linkers/12'>RuvC and HNH</scene> are involved in endonuclease activity. RuvC uses two manganese to cleave the non-target DNA through manganese coordinating with the phosphate backbone and aspartic acid residues. A histidine then acts as a base to create a hydroxide nucleophile that attacks the phosphate bond and cleaves the non-target DNA. The binding of the RuvC to the target DNA changes the conformation of a linker protein region between the RuvC domain and the HNH domain. The conformational change of the linker bings the HNH domain close enough to the target DNA to cut the DNA. The HNH follows a similar mechanism as to RuvC using a histidine base to create a hydroxide ion nucleophile that attacks the phosphate bond. This is modeled as manganese however, it is often magnesium in cell <ref name="Cas9" />.<br />
</structuresection><br />
==References==<br />
<br />
<references /></div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3644186Sandbox Reserved 17502022-10-17T16:39:37Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections. CRISPR is a form of acquired immunity used by bacteria. CRISPR stands for clustered regularly interspaced short palindromic repeats because the bacterial genome includes genetic sequences clustered together from bacteriophages of previous infections that are used as by Cas9 to cut viral DNA. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA, rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single-stranded guide RNA (sgRNA) to complimentarily bind the target DNA that will create a double stranded DNA cut in the proper location. The target DNA must also have a PAM sequence to bind for Cas9 to cut target DNA. The PAM sequence stands for protospacer adjacent motif and is downstream from the cut site of the nuclease. The PAM sequence acts as a two-factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. <br />
<br />
The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the <scene name='92/925538/Lobes_and_linkers/16'>REC lobe</scene> (residues 41–425) and <scene name='92/925538/Lobes_and_linkers/15'>NUC lobe</scene> (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WED, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The NUC lobe stands for the nuclease lobe and each of these domains are a part of the protein that cuts the target DNA <ref>PMID:15596446</ref><ref>PMID:24634220</ref><ref>PMID:24529477</ref>. The REC lobe stands for the recognition lobe is responsible for recognizing the target DNA present causing a conformational change in the HNH domain locking the HNH domain into the cleavage site <ref>PMID:30555184</ref>. Cas9 has four main mechanisms that are important for successful cleavage, including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe and bridge helix interacts with the seed region of the <scene name='92/925538/Lobes_and_linkers/17'>sgRNA</scene> (C13-C20). The seed region is in the <scene name='92/925538/Lobes_and_linkers/8'>A-form conformation</scene>, so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the <scene name='92/925538/Lobes_and_linkers/18'>phosphate backbone</scene>. The target DNA binds to the <scene name='92/925538/Lobes_and_linkers/9'>REC lobe and RuvC domain</scene> for the proper conformation for base paring between the target DNA and sgRNA<ref name="Cas9" />.<br />
== Recognition of the PAM sequence ==<br />
For the recognition of the <scene name='92/925538/Lobes_and_linkers/19'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through hydrogen bonds as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. This PAM sequence is differnt that other PAM sequences like the one found in SpCas9 (5'-NGG-3'). The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.<br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the <scene name='92/925538/Lobes_and_linkers/20'>REC lobe and WED domain</scene>. The WED contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat: anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="Cas9" />.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, <scene name='92/925538/Lobes_and_linkers/12'>RuvC and HNH</scene> are in endonuclease activity. RuvC uses two manganese to cleave the non-target DNA through manganese coordinating with the phosphate backbone and aspartic acid residues. A histidine then acts as a base to create a hydroxide nucleophile that attacks the phosphate bond and cleaves the non-target DNA. The binding of the RuvC to the target DNA changes the conformation of a linker protein region between the RuvC domain and the HNH domain. The conformational change of the linker bings the HNH domain close enough to the target DNA to cut the DNA. The HNH follows a similar mechanism as to RuvC using a histidine base to create a hydroxide ion nucleophile that attacks the phosphate bond. This is modeled as manganese however, it is often magnesium in cell <ref name="Cas9" />.<br />
</structuresection><br />
==References==<br />
<br />
<references /></div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3644185Sandbox Reserved 17502022-10-17T16:18:45Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections. CRISPR is a form of acquired immunity used by bacteria. CRISPR stands for clustered regularly interspaced short palindromic repeats because the bacterial genome includes genetic sequences clustered together from bacteriophages of previous infections that are used as by Cas9 to cut viral DNA. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA, rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single-stranded guide RNA (sgRNA) to complimentarily bind the target DNA that will create a double stranded DNA cut in the proper location. The target DNA must also have a PAM sequence to bind for Cas9 to cut target DNA. The PAM sequence stands for protospacer adjacent motif and is downstream from the cut site of the nuclease. The PAM sequence acts as a two-factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. <br />
<br />
The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the <scene name='92/925538/Lobes_and_linkers/16'>REC lobe</scene> (residues 41–425) and <scene name='92/925538/Lobes_and_linkers/15'>NUC lobe</scene> (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WED, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The NUC lobe stands for the nuclease lobe and each of these domains are a part of the protein that cuts the target DNA <ref>PMID:15596446</ref><ref>PMID:24634220</ref><ref>PMID:24529477</ref>. The REC lobe stands for the recognition lobe is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site <ref>PMID:30555184</ref>. Cas9 has four main mechanisms that are important for successful cleavage, including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe and bridge helix interacts with the seed region of the <scene name='92/925538/Lobes_and_linkers/17'>sgRNA</scene> (C13-C20). The seed region is in the <scene name='92/925538/Lobes_and_linkers/8'>A-form conformation</scene>, so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the <scene name='92/925538/Lobes_and_linkers/18'>phosphate backbone</scene>. The target DNA binds to the <scene name='92/925538/Lobes_and_linkers/9'>REC lobe and RuvC domain</scene> for the proper conformation for base paring between the target DNA and sgRNA<ref name="Cas9" />.<br />
== Recognition of the PAM sequence ==<br />
For the recognition of the <scene name='92/925538/Lobes_and_linkers/19'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through hydrogen bonds as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. This PAM sequence is differnt that other PAM sequences like the one found in SpCas9 (5'-NGG-3'). The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.<br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the <scene name='92/925538/Lobes_and_linkers/20'>REC lobe and WED domain</scene>. The WED contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat: anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="Cas9" />.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, <scene name='92/925538/Lobes_and_linkers/12'>RuvC and HNH</scene> are in endonuclease activity. RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This is modeled as manganese however, it is often magnesium in cell. The magnesium allows a histidine to become a general base and cleave the target DNA. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA <ref name="Cas9" />.<br />
</structuresection><br />
==References==<br />
<br />
<references /></div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3644184Sandbox Reserved 17502022-10-17T16:18:19Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections. CRISPR is a form of acquired immunity used by bacteria. CRISPR stands for clustered regularly interspaced short palindromic repeats because the bacterial genome includes genetic sequences clustered together from bacteriophages of previous infections that are used as by Cas9 to cut viral DNA. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA, rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single-stranded guide RNA (sgRNA) to complimentarily bind the target DNA that will create a double stranded DNA cut in the proper location. The target DNA must also have a PAM sequence to bind for Cas9 to cut target DNA. The PAM sequence stands for protospacer adjacent motif and is downstream from the cut site of the nuclease. The PAM sequence acts as a two-factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. <br />
The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the <scene name='92/925538/Lobes_and_linkers/16'>REC lobe</scene> (residues 41–425) and <scene name='92/925538/Lobes_and_linkers/15'>NUC lobe</scene> (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WED, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The NUC lobe stands for the nuclease lobe and each of these domains are a part of the protein that cuts the target DNA <ref>PMID:15596446</ref><ref>PMID:24634220</ref><ref>PMID:24529477</ref>. The REC lobe stands for the recognition lobe is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site <ref>PMID:30555184</ref>. Cas9 has four main mechanisms that are important for successful cleavage, including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe and bridge helix interacts with the seed region of the <scene name='92/925538/Lobes_and_linkers/17'>sgRNA</scene> (C13-C20). The seed region is in the <scene name='92/925538/Lobes_and_linkers/8'>A-form conformation</scene>, so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the <scene name='92/925538/Lobes_and_linkers/18'>phosphate backbone</scene>. The target DNA binds to the <scene name='92/925538/Lobes_and_linkers/9'>REC lobe and RuvC domain</scene> for the proper conformation for base paring between the target DNA and sgRNA<ref name="Cas9" />.<br />
== Recognition of the PAM sequence ==<br />
For the recognition of the <scene name='92/925538/Lobes_and_linkers/19'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through hydrogen bonds as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. This PAM sequence is differnt that other PAM sequences like the one found in SpCas9 (5'-NGG-3'). The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.<br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the <scene name='92/925538/Lobes_and_linkers/20'>REC lobe and WED domain</scene>. The WED contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat: anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="Cas9" />.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, <scene name='92/925538/Lobes_and_linkers/12'>RuvC and HNH</scene> are in endonuclease activity. RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This is modeled as manganese however, it is often magnesium in cell. The magnesium allows a histidine to become a general base and cleave the target DNA. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA <ref name="Cas9" />.<br />
</structuresection><br />
==References==<br />
<br />
<references /></div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3644110Sandbox Reserved 17502022-10-11T12:05:02Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA, rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single-stranded guide RNA (sgRNA) to bind the target DNA that will be cut. Cas9 utilizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double-stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind for Cas9 to cut target DNA. The PAM sequence acts as a two-factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the REC lobe (residues 41–425) and NUC lobe (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The <scene name='92/925538/Lobes_and_linkers/15'>NUC lobe</scene> contains RuvC, HNH, WED, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The <scene name='92/925538/Lobes_and_linkers/16'>REC lobe</scene> is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site <ref>PMID:30555184</ref>. The RuvC nuclease domain (residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the [[sgRNA]] <ref name="sgRNA">PMID:24529477</ref>. The HNH domain (residues 520–628) cleaves the target strand of DNA bound to the sgRNA <ref name="sgRNA" />. The WED domain (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of a twisted five-stranded beta sheet flanked by four alpha helices <ref>PMID:15596446</ref>. The PI domain (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA <ref>PMID:24634220</ref>. There are also two linker domains, L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH <ref name="Cas9" />. Furthermore, there is a phosphate lock loop (scene of loop) (residues 775–787) that connect the WED and RuvC domains. Cas9 has four main mechanisms that are important for successful cleavage, including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe and bridge helix interacts with the seed region of the <scene name='92/925538/Lobes_and_linkers/17'>sgRNA</scene> (C13-C20). The seed region is in the <scene name='92/925538/Lobes_and_linkers/8'>A-form conformation</scene>, so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the <scene name='92/925538/Lobes_and_linkers/18'>phosphate backbone</scene>. The target DNA binds to the <scene name='92/925538/Lobes_and_linkers/9'>REC lobe and RuvC domain</scene> for the proper conformation for base paring between the target DNA and sgRNA<ref name="Cas9" />.<br />
== Recognition of the PAM sequence ==<br />
For the recognition of the <scene name='92/925538/Lobes_and_linkers/19'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through hydrogen bonds as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. This PAM sequence is differnt that other PAM sequences like the one found in SpCas9 (5'-NGG-3'). The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.<br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the <scene name='92/925538/Lobes_and_linkers/20'>REC lobe and WED domain</scene>. The WED contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat: anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="Cas9" />.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, <scene name='92/925538/Lobes_and_linkers/12'>RuvC and HNH</scene> are in endonuclease activity. RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This is modeled as manganese however, it is often magnesium in cell. The magnesium allows a histidine to become a general base and cleave the target DNA. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA <ref name="Cas9" />.<br />
</structuresection><br />
==References==<br />
<br />
<references /></div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3644108Sandbox Reserved 17502022-10-11T11:57:44Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA, rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single-stranded guide RNA (sgRNA) to bind the target DNA that will be cut. Cas9 utilizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double-stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind for Cas9 to cut target DNA. The PAM sequence acts as a two-factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the REC lobe (residues 41–425) and NUC lobe (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The <scene name='92/925538/Lobes_and_linkers/15'>NUC lobe</scene> contains RuvC, HNH, WED, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The <scene name='92/925538/Lobes_and_linkers/16'>REC lobe</scene> is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site <ref>PMID:30555184</ref>. The RuvC nuclease domain (residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the [[sgRNA]] <ref name="sgRNA">PMID:24529477</ref>. The HNH domain (residues 520–628) cleaves the target strand of DNA bound to the sgRNA <ref name="sgRNA" />. The WED domain (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of a twisted five-stranded beta sheet flanked by four alpha helices <ref>PMID:15596446</ref>. The PI domain (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA <ref>PMID:24634220</ref>. There are also two linker domains, L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH <ref name="Cas9" />. Furthermore, there is a phosphate lock loop (scene of loop) (residues 775–787) that connect the WED and RuvC domains. Cas9 has four main mechanisms that are important for successful cleavage, including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe and bridge helix interacts with the seed region of the <scene name='92/925538/Lobes_and_linkers/17'>sgRNA</scene> (C13-C20). The seed region is in the <scene name='92/925538/Lobes_and_linkers/8'>A-form conformation</scene>, so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the <scene name='92/925538/Lobes_and_linkers/18'>phosphate backbone</scene>. The target DNA binds to the <scene name='92/925538/Lobes_and_linkers/9'>REC lobe and RuvC domain</scene> for the proper conformation for base paring between the target DNA and sgRNA<ref name="Cas9" />.<br />
== Recognition of the PAM sequence ==<br />
For the recognition of the <scene name='92/925538/Lobes_and_linkers/19'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds (scene of bonds) as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.<br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the <scene name='92/925538/Lobes_and_linkers/20'>REC lobe and WED domain</scene>. The WED contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat: anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="Cas9" />.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, <scene name='92/925538/Lobes_and_linkers/12'>RuvC and HNH</scene> are in endonuclease activity (Scene). RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This is modeled as manganese however, it is often magnesium in cell. The magnesium allows a histidine to become a general base and cleave the target DNA. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA <ref name="Cas9" />.<br />
</structuresection><br />
==References==<br />
<br />
<references /></div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3644040Sandbox Reserved 17502022-10-10T23:14:07Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA, rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single-stranded guide RNA (sgRNA) to bind the target DNA that will be cut. Cas9 utilizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double-stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind to Cas9 to be cut. The PAM sequence acts as a two-factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the REC lobe (residues 41–425) and NUC lobe (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The <scene name='92/925538/Lobes_and_linkers/15'>NUC lobe</scene> contains RuvC, HNH, WED, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The <scene name='92/925538/Lobes_and_linkers/16'>REC lobe</scene> is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site <ref>PMID:30555184</ref>. The RuvC nuclease domain (residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the [[sgRNA]] <ref name="sgRNA">PMID:24529477</ref>. The HNH domain (residues 520–628) cleaves the target strand of DNA bound to the sgRNA <ref name="sgRNA" />. The WED domain (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of a twisted five-stranded beta sheet flanked by four alpha helices <ref>PMID:15596446</ref>. The PI domain (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA <ref>PMID:24634220</ref>. There are also two linker domains, L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH <ref name="Cas9" />. Furthermore, there is a phosphate lock loop (scene of loop) (residues 775–787) that connect the WED and RuvC domains. Cas9 has four main mechanisms that are important for successful cleavage, including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe and bridge helix interacts with the seed region of the <scene name='92/925538/Lobes_and_linkers/17'>sgRNA</scene> (C13-C20). The seed region is in the <scene name='92/925538/Lobes_and_linkers/8'>A-form conformation</scene>, so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the <scene name='92/925538/Lobes_and_linkers/18'>phosphate backbone</scene>. The target DNA binds to the <scene name='92/925538/Lobes_and_linkers/9'>REC lobe and RuvC domain</scene> for the proper conformation for base paring between the target DNA and sgRNA<ref name="Cas9" />.<br />
== Recognition of the PAM sequence ==<br />
For the recognition of the <scene name='92/925538/Lobes_and_linkers/19'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds (scene of bonds) as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.<br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the <scene name='92/925538/Lobes_and_linkers/20'>REC lobe and WED domain</scene>. The WED contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat: anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="Cas9" />.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, <scene name='92/925538/Lobes_and_linkers/12'>RuvC and HNH</scene> are in endonuclease activity (Scene). RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This is modeled as manganese however, it is often magnesium in cell. The magnesium allows a histidine to become a general base and cleave the target DNA. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA <ref name="Cas9" />.<br />
</structuresection><br />
==References==<br />
<br />
<references /></div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3644038Sandbox Reserved 17502022-10-10T20:45:48Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA, rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single-stranded guide RNA (sgRNA) to bind the target DNA that will be cut. Cas9 utilizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double-stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind to Cas9 to be cut. The PAM sequence acts as a two-factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the REC lobe (residues 41–425) and NUC lobe (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The <scene name='92/925538/Lobes_and_linkers/15'>NUC lobe</scene> contains RuvC, HNH, WED, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The <scene name='92/925538/Lobes_and_linkers/16'>REC lobe</scene> is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site <ref>PMID:30555184</ref>. The RuvC nuclease domain (residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the [[sgRNA]] <ref name="sgRNA">PMID:24529477</ref>. The HNH domain (residues 520–628) cleaves the target strand of DNA bound to the sgRNA <ref name="sgRNA" />. The WED domain (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of a twisted five-stranded beta sheet flanked by four alpha helices <ref>PMID:15596446</ref>. The PI domain (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA <ref>PMID:24634220</ref>. There are also two linker domains, L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH <ref name="Cas9" />. Furthermore, there is a phosphate lock loop (scene of loop) (residues 775–787) that connect the WED and RuvC domains. Cas9 has four main mechanisms that are important for successful cleavage, including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe and bridge helix interacts with the seed region of the <scene name='92/925538/Lobes_and_linkers/17'>sgRNA</scene> (C13-C20). The seed region is in the <scene name='92/925538/Lobes_and_linkers/8'>A-form conformation</scene>, so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the <scene name='92/925538/Lobes_and_linkers/18'>phosphate backbone</scene>. The target DNA binds to the <scene name='92/925538/Lobes_and_linkers/9'>REC lobe and RuvC domain</scene> for the proper conformation for base paring between the target DNA and sgRNA<ref name="Cas9" />.<br />
== Recognition of the PAM sequence ==<br />
For the recognition of the <scene name='92/925538/Lobes_and_linkers/19'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds (scene of bonds) as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.<br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the <scene name='92/925538/Lobes_and_linkers/19'>REC lobe and WED domain</scene>. The WED contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat: anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="Cas9" />.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, <scene name='92/925538/Lobes_and_linkers/12'>RuvC and HNH</scene> are in endonuclease activity (Scene). RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This is modeled as manganese however, it is often magnesium in cell. The magnesium allows a histidine to become a general base and cleave the target DNA. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA <ref name="Cas9" />.<br />
</structuresection><br />
==References==<br />
<br />
<references /></div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3644037Sandbox Reserved 17502022-10-10T20:34:18Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA, rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single-stranded guide RNA (sgRNA) to bind the target DNA that will be cut. Cas9 utilizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double-stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind to Cas9 to be cut. The PAM sequence acts as a two-factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the REC lobe (residues 41–425) and NUC lobe (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The <scene name='92/925538/Lobes_and_linkers/15'>NUC lobe</scene> contains RuvC, HNH, WED, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The <scene name='92/925538/Lobes_and_linkers/16'>REC lobe</scene> is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site <ref>PMID:30555184</ref>. The RuvC nuclease domain (residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the [[sgRNA]] <ref name="sgRNA">PMID:24529477</ref>. The HNH domain (residues 520–628) cleaves the target strand of DNA bound to the sgRNA <ref name="sgRNA" />. The WED domain (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of a twisted five-stranded beta sheet flanked by four alpha helices <ref>PMID:15596446</ref>. The PI domain (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA <ref>PMID:24634220</ref>. There are also two linker domains, L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH <ref name="Cas9" />. Furthermore, there is a phosphate lock loop (scene of loop) (residues 775–787) that connect the WED and RuvC domains. Cas9 has four main mechanisms that are important for successful cleavage, including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe and bridge helix interacts with the seed region of the <scene name='92/925538/Lobes_and_linkers/17'>sgRNA</scene> (C13-C20). The seed region is in the <scene name='92/925538/Lobes_and_linkers/8'>A-form conformation</scene>, so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the <scene name='92/925538/Lobes_and_linkers/18'>phosphate backbone</scene>. The target DNA binds to the <scene name='92/925538/Lobes_and_linkers/9'>REC lobe and RuvC domain</scene> for the proper conformation for base paring between the target DNA and sgRNA<ref name="Cas9" />.<br />
== Recognition of the PAM sequence ==<br />
For the recognition of the <scene name='92/925538/Lobes_and_linkers/19'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds (scene of bonds) as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.<br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the <scene name='92/925538/Lobes_and_linkers/19'>REC lobe and WED domain</scene>. The WED contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat: anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="Cas9" />.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, <scene name='92/925538/Lobes_and_linkers/12'>RuvC and HNH</scene> are in endonuclease activity (Scene). RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA <ref name="Cas9" />.<br />
</structuresection><br />
==References==<br />
<br />
<references /></div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3644036Sandbox Reserved 17502022-10-10T20:30:38Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single stranded guide RNA (sgRNA) to bind the target DNA that will be cut. Cas9 utilizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind to Cas9 to be cut. The PAM sequence acts as a two factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the REC lobe (residues 41–425) and NUC lobe (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The <scene name='92/925538/Lobes_and_linkers/15'>NUC lobe</scene> contains RuvC, HNH, WED, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The <scene name='92/925538/Lobes_and_linkers/16'>REC lobe</scene> is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site <ref>PMID:30555184</ref>. The RuvC nuclease domain (residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the [[sgRNA]] <ref name="sgRNA">PMID:24529477</ref>. The HNH domain (residues 520–628) cleaves the target strand of DNA bound to the sgRNA <ref name="sgRNA" />. The WED domain (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of twisted five-stranded beta sheet flanked by four alpha helices <ref>PMID:15596446</ref>. The PI domain (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA <ref>PMID:24634220</ref>. There are also two linker domains, L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH <ref name="Cas9" />. Furthermore, there is a phosphate lock loop (scene of loop) (residues 775–787) that connect the WED and RuvC domains. Cas9 has four main mechanisms that are important for successful cleavage including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is a the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe and bridge helix interacts with the seed region of the <scene name='92/925538/Lobes_and_linkers/17'>sgRNA</scene> (C13-C20). The seed region is in the <scene name='92/925538/Lobes_and_linkers/8'>A-form conformation</scene> so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the <scene name='92/925538/Lobes_and_linkers/18'>phosphate backbone</scene>. The target DNA binds to the <scene name='92/925538/Lobes_and_linkers/9'>REC lobe and RuvC domain</scene> for the proper conformation for base paring between the target DNA and sgRNA<ref name="Cas9" />.<br />
== Recognition of the PAM sequence ==<br />
For the recognition of the <scene name='92/925538/Lobes_and_linkers/19'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds (scene of bonds) as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.<br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the <scene name='92/925538/Lobes_and_linkers/19'>REC lobe and WED domain</scene>. The WED contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat: anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="Cas9" />.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, <scene name='92/925538/Lobes_and_linkers/12'>RuvC and HNH</scene> are in endonuclease activity (Scene). RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA <ref name="Cas9" />.<br />
</structuresection><br />
==References==<br />
<br />
<references /></div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3644035Sandbox Reserved 17502022-10-10T20:29:24Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single stranded guide RNA (sgRNA) to bind the target DNA that will be cut. Cas9 utalizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind to Cas9 to be cut. The PAM sequence acts as a two factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the REC lobe (residues 41–425) and NUC lobe (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The <scene name='92/925538/Lobes_and_linkers/15'>NUC lobe</scene> contains RuvC, HNH, WED, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The <scene name='92/925538/Lobes_and_linkers/16'>REC lobe</scene> is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site <ref>PMID:30555184</ref>. The RuvC nuclease domain (residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the [[sgRNA]] <ref name="sgRNA">PMID:24529477</ref>. The HNH domain (residues 520–628) cleaves the target strand of DNA bound to the sgRNA <ref name="sgRNA" />. The WEB domain (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of twisted five-stranded beta sheet flanked by four alpha helices <ref>PMID:15596446</ref>. The PI domain (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA <ref>PMID:24634220</ref>. There are also two linker domains, L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH <ref name="Cas9" />. Furthermore, there is a phosphate lock loop (scene of loop) (residues 775–787) that connect the WEB and RuvC domains. Cas9 has four main mechanisms that are important for successful cleavage including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is a the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe and bridge helix interacts with the seed region of the <scene name='92/925538/Lobes_and_linkers/17'>sgRNA</scene> (C13-C20). The seed region is in the <scene name='92/925538/Lobes_and_linkers/8'>A-form conformation</scene> so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the <scene name='92/925538/Lobes_and_linkers/18'>phosphate backbone</scene>. The target DNA binds to the <scene name='92/925538/Lobes_and_linkers/9'>REC lobe and RuvC domain</scene> for the proper conformation for base paring between the target DNA and sgRNA<ref name="Cas9" />.<br />
== Recognition of the PAM sequence ==<br />
For the recognition of the <scene name='92/925538/Lobes_and_linkers/19'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds (scene of bonds) as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.<br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the <scene name='92/925538/Lobes_and_linkers/19'>REC lobe and WED domain</scene>. The WED contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat:anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="Cas9" />.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, <scene name='92/925538/Lobes_and_linkers/12'>RuvC and HNH</scene> are in endonuclease activity (Scene). RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA <ref name="Cas9" />.<br />
</structuresection><br />
==References==<br />
<br />
<references /></div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3644031Sandbox Reserved 17502022-10-10T20:14:34Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single stranded guide RNA (sgRNA) to bind the target DNA that will be cut. Cas9 utalizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind to Cas9 to be cut. The PAM sequence acts as a two factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the REC lobe (residues 41–425) and NUC lobe (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The <scene name='92/925538/Lobes_and_linkers/15'>NUC lobe</scene> contains RuvC, HNH, WED, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The <scene name='92/925538/Lobes_and_linkers/16'>REC lobe</scene> is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site <ref>PMID:30555184</ref>. The RuvC nuclease domain (residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the [[sgRNA]] <ref name="sgRNA">PMID:24529477</ref>. The HNH domain (residues 520–628) cleaves the target strand of DNA bound to the sgRNA <ref name="sgRNA" />. The WEB domain (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of twisted five-stranded beta sheet flanked by four alpha helices <ref>PMID:15596446</ref>. The PI domain (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA <ref>PMID:24634220</ref>. There are also two linker domains, L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH <ref name="Cas9" />. Furthermore, there is a phosphate lock loop (scene of loop) (residues 775–787) that connect the WEB and RuvC domains. Cas9 has four main mechanisms that are important for successful cleavage including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is a the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe and bridge helix interacts with the seed region of the <scene name='92/925538/Lobes_and_linkers/17'>sgRNA</scene> (C13-C20). The seed region is in the <scene name='92/925538/Lobes_and_linkers/8'>A-form conformation</scene> so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the <scene name='92/925538/Lobes_and_linkers/18'>phosphate backbone</scene>. The target DNA binds to the <scene name='92/925538/Lobes_and_linkers/9'>REC lobe and RuvC domain</scene> for the proper conformation for base paring between the target DNA and sgRNA<ref name="Cas9" />.<br />
== Recognition of the PAM sequence ==<br />
For the recognition of the <scene name='92/925538/Lobes_and_linkers/10'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds (scene of bonds) as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.<br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the <scene name='92/925538/Lobes_and_linkers/11'>REC lobe and WED domain</scene>. The <scene name='92/925538/Lobes_and_linkers/11'>WED domain</scene> contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat:anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="Cas9" />.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, <scene name='92/925538/Lobes_and_linkers/12'>RuvC and HNH</scene> are in endonuclease activity (Scene). RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA <ref name="Cas9" />.<br />
</structuresection><br />
==References==<br />
<br />
<references /></div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3644030Sandbox Reserved 17502022-10-10T20:11:31Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single stranded guide RNA (sgRNA) to bind the target DNA that will be cut. Cas9 utalizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind to Cas9 to be cut. The PAM sequence acts as a two factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the REC lobe (residues 41–425) and NUC lobe (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The <scene name='92/925538/Lobes_and_linkers/15'>NUC lobe</scene> contains RuvC, HNH, WED, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The <scene name='92/925538/Lobes_and_linkers/16'>REC lobe</scene> is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site <ref>PMID:30555184</ref>. The RuvC nuclease domain (residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the [[sgRNA]] <ref name="sgRNA">PMID:24529477</ref>. The HNH domain (residues 520–628) cleaves the target strand of DNA bound to the sgRNA <ref name="sgRNA" />. The WEB domain (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of twisted five-stranded beta sheet flanked by four alpha helices <ref>PMID:15596446</ref>. The PI domain (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA <ref>PMID:24634220</ref>. There are also two linker domains, L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH <ref name="Cas9" />. Furthermore, there is a phosphate lock loop (scene of loop) (residues 775–787) that connect the WEB and RuvC domains. Cas9 has four main mechanisms that are important for successful cleavage including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is a the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe and bridge helix interacts with the seed region of the <scene name='92/925538/Lobes_and_linkers/17'>sgRNA</scene> (C13-C20). The seed region is in the <scene name='92/925538/Lobes_and_linkers/8'>A-form conformation</scene> so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the <scene name='92/925538/Lobes_and_linkers/18'>phosphate backbone</scene>. The target DNA binds to the REC loop and RuvC domain for the proper conformation for base paring between the <scene name='92/925538/Lobes_and_linkers/9'>Target DNA and sgRNA</scene><ref name="Cas9" />.<br />
== Recognition of the PAM sequence ==<br />
For the recognition of the <scene name='92/925538/Lobes_and_linkers/10'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds (scene of bonds) as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.<br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the <scene name='92/925538/Lobes_and_linkers/11'>REC lobe and WED domain</scene>. The <scene name='92/925538/Lobes_and_linkers/11'>WED domain</scene> contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat:anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="Cas9" />.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, <scene name='92/925538/Lobes_and_linkers/12'>RuvC and HNH</scene> are in endonuclease activity (Scene). RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA <ref name="Cas9" />.<br />
</structuresection><br />
==References==<br />
<br />
<references /></div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3644028Sandbox Reserved 17502022-10-10T19:56:41Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single stranded guide RNA (sgRNA) to bind the target DNA that will be cut. Cas9 utalizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind to Cas9 to be cut. The PAM sequence acts as a two factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the REC lobe (residues 41–425) and NUC lobe (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The <scene name='92/925538/Lobes_and_linkers/15'>NUC lobe</scene> contains RuvC, HNH, WED, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The <scene name='92/925538/Lobes_and_linkers/16'>REC lobe</scene> is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site <ref>PMID:30555184</ref>. The RuvC nuclease domain (residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the [[sgRNA]] <ref name="sgRNA">PMID:24529477</ref>. The HNH domain (residues 520–628) cleaves the target strand of DNA bound to the sgRNA <ref name="sgRNA" />. The WEB domain (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of twisted five-stranded beta sheet flanked by four alpha helices <ref>PMID:15596446</ref>. The PI domain (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA <ref>PMID:24634220</ref>. There are also two linker domains, L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH <ref name="Cas9" />. Furthermore, there is a phosphate lock loop (scene of loop) (residues 775–787) that connect the WEB and RuvC domains. Cas9 has four main mechanisms that are important for successful cleavage including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is a the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe and bridge helix interacts with the seed region of the <scene name='92/925538/Lobes_and_linkers/5'>sgRNA</scene> (C13-C20). The seed region is in the <scene name='92/925538/Lobes_and_linkers/8'>A-form conformation</scene> so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the <scene name='92/925538/Lobes_and_linkers/7'>phosphate backbone</scene>. The target DNA binds to the REC loop and RuvC domain for the proper conformation for base paring between the <scene name='92/925538/Lobes_and_linkers/9'>Target DNA and sgRNA</scene><ref name="Cas9" />.<br />
== Recognition of the PAM sequence ==<br />
For the recognition of the <scene name='92/925538/Lobes_and_linkers/10'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds (scene of bonds) as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.<br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the <scene name='92/925538/Lobes_and_linkers/11'>REC lobe and WED domain</scene>. The <scene name='92/925538/Lobes_and_linkers/11'>WED domain</scene> contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat:anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="Cas9" />.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, <scene name='92/925538/Lobes_and_linkers/12'>RuvC and HNH</scene> are in endonuclease activity (Scene). RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA <ref name="Cas9" />.<br />
</structuresection><br />
==References==<br />
<br />
<references /></div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3644027Sandbox Reserved 17502022-10-10T19:54:02Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single stranded guide RNA (sgRNA) to bind the target DNA that will be cut. Cas9 utalizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind to Cas9 to be cut. The PAM sequence acts as a two factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the REC lobe (residues 41–425) and NUC lobe (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The <scene name='92/925538/Lobes_and_linkers/15'>NUC lobe</scene> contains RuvC, HNH, WED, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The <scene name='92/925538/Lobes_and_linkers/14'>REC lobe</scene> is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site <ref>PMID:30555184</ref>. The RuvC nuclease domain (residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the [[sgRNA]] <ref name="sgRNA">PMID:24529477</ref>. The HNH domain (residues 520–628) cleaves the target strand of DNA bound to the sgRNA <ref name="sgRNA" />. The WEB domain (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of twisted five-stranded beta sheet flanked by four alpha helices <ref>PMID:15596446</ref>. The PI domain (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA <ref>PMID:24634220</ref>. There are also two linker domains, L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH <ref name="Cas9" />. Furthermore, there is a phosphate lock loop (scene of loop) (residues 775–787) that connect the WEB and RuvC domains. Cas9 has four main mechanisms that are important for successful cleavage including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is a the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe and bridge helix interacts with the seed region of the <scene name='92/925538/Lobes_and_linkers/5'>sgRNA</scene> (C13-C20). The seed region is in the <scene name='92/925538/Lobes_and_linkers/8'>A-form conformation</scene> so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the <scene name='92/925538/Lobes_and_linkers/7'>phosphate backbone</scene>. The target DNA binds to the REC loop and RuvC domain for the proper conformation for base paring between the <scene name='92/925538/Lobes_and_linkers/9'>Target DNA and sgRNA</scene><ref name="Cas9" />.<br />
== Recognition of the PAM sequence ==<br />
For the recognition of the <scene name='92/925538/Lobes_and_linkers/10'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds (scene of bonds) as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.<br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the <scene name='92/925538/Lobes_and_linkers/11'>REC lobe and WED domain</scene>. The <scene name='92/925538/Lobes_and_linkers/11'>WED domain</scene> contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat:anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="Cas9" />.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, <scene name='92/925538/Lobes_and_linkers/12'>RuvC and HNH</scene> are in endonuclease activity (Scene). RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA <ref name="Cas9" />.<br />
</structuresection><br />
==References==<br />
<br />
<references /></div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3644024Sandbox Reserved 17502022-10-10T19:37:35Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single stranded guide RNA (sgRNA) to bind the target DNA that will be cut. Cas9 utalizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind to Cas9 to be cut. The PAM sequence acts as a two factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the REC lobe (residues 41–425) and NUC lobe (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WED, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The <scene name='92/925538/Lobes_and_linkers/14'>REC lobe</scene> is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site <ref>PMID:30555184</ref>. The RuvC nuclease domain (residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the [[sgRNA]] <ref name="sgRNA">PMID:24529477</ref>. The HNH domain (residues 520–628) cleaves the target strand of DNA bound to the sgRNA <ref name="sgRNA" />. The WEB domain (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of twisted five-stranded beta sheet flanked by four alpha helices <ref>PMID:15596446</ref>. The PI domain (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA <ref>PMID:24634220</ref>. There are also two linker domains, L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH <ref name="Cas9" />. Furthermore, there is a phosphate lock loop (scene of loop) (residues 775–787) that connect the WEB and RuvC domains. Cas9 has four main mechanisms that are important for successful cleavage including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is a the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe and bridge helix interacts with the seed region of the <scene name='92/925538/Lobes_and_linkers/5'>sgRNA</scene> (C13-C20). The seed region is in the <scene name='92/925538/Lobes_and_linkers/8'>A-form conformation</scene> so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the <scene name='92/925538/Lobes_and_linkers/7'>phosphate backbone</scene>. The target DNA binds to the REC loop and RuvC domain for the proper conformation for base paring between the <scene name='92/925538/Lobes_and_linkers/9'>Target DNA and sgRNA</scene><ref name="Cas9" />.<br />
== Recognition of the PAM sequence ==<br />
For the recognition of the <scene name='92/925538/Lobes_and_linkers/10'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds (scene of bonds) as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.<br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the <scene name='92/925538/Lobes_and_linkers/11'>REC lobe and WED domain</scene>. The <scene name='92/925538/Lobes_and_linkers/11'>WED domain</scene> contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat:anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="Cas9" />.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, <scene name='92/925538/Lobes_and_linkers/12'>RuvC and HNH</scene> are in endonuclease activity (Scene). RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA <ref name="Cas9" />.<br />
</structuresection><br />
==References==<br />
<br />
<references /></div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3644023Sandbox Reserved 17502022-10-10T19:35:03Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single stranded guide RNA (sgRNA) to bind the target DNA that will be cut. Cas9 utalizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind to Cas9 to be cut. The PAM sequence acts as a two factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the REC lobe (residues 41–425) and NUC lobe (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WED, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The <scene name='92/925538/Lobes_and_linkers/14'>REC lobe</scene> is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site <ref>PMID:30555184</ref>. The RuvC nuclease domain (Scene of domain)(residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the [[sgRNA]] <ref name="sgRNA">PMID:24529477</ref>. The HNH domain (scene of domain) (residues 520–628) cleaves the target strand of DNA bound to the sgRNA <ref name="sgRNA" />. The WEB domain (Scene of domain) (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of twisted five-stranded beta sheet flanked by four alpha helices <ref>PMID:15596446</ref>. The PI domain (scene of domain) (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA <ref>PMID:24634220</ref>. There are also two linker domains (scene of domain), L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH <ref name="Cas9" />. Furthermore, there is a phosphate lock loop (scene of loop) (residues 775–787) that connect the WEB and RuvC domains. Cas9 has four main mechanisms that are important for successful cleavage including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is a the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe and bridge helix interacts with the seed region of the <scene name='92/925538/Lobes_and_linkers/5'>sgRNA</scene> (C13-C20). The seed region is in the <scene name='92/925538/Lobes_and_linkers/8'>A-form conformation</scene> so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the <scene name='92/925538/Lobes_and_linkers/7'>phosphate backbone</scene>. The target DNA binds to the REC loop and RuvC domain for the proper conformation for base paring between the <scene name='92/925538/Lobes_and_linkers/9'>Target DNA and sgRNA</scene><ref name="Cas9" />.<br />
== Recognition of the PAM sequence ==<br />
For the recognition of the <scene name='92/925538/Lobes_and_linkers/10'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds (scene of bonds) as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.<br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the <scene name='92/925538/Lobes_and_linkers/11'>REC lobe and WED domain</scene>. The <scene name='92/925538/Lobes_and_linkers/11'>WED domain</scene> contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat:anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="Cas9" />.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, <scene name='92/925538/Lobes_and_linkers/12'>RuvC and HNH</scene> are in endonuclease activity (Scene). RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA <ref name="Cas9" />.<br />
</structuresection><br />
==References==<br />
<br />
<references /></div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3644022Sandbox Reserved 17502022-10-10T19:31:59Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single stranded guide RNA (sgRNA) to bind the target DNA that will be cut. Cas9 utalizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind to Cas9 to be cut. The PAM sequence acts as a two factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the REC lobe (residues 41–425) and NUC lobe (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WED, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The <scene name='92/925538/Lobes_and_linkers/13'>REC lobe</scene> is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site <ref>PMID:30555184</ref>. The RuvC nuclease domain (Scene of domain)(residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the [[sgRNA]] <ref name="sgRNA">PMID:24529477</ref>. The HNH domain (scene of domain) (residues 520–628) cleaves the target strand of DNA bound to the sgRNA <ref name="sgRNA" />. The WEB domain (Scene of domain) (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of twisted five-stranded beta sheet flanked by four alpha helices <ref>PMID:15596446</ref>. The PI domain (scene of domain) (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA <ref>PMID:24634220</ref>. There are also two linker domains (scene of domain), L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH <ref name="Cas9" />. Furthermore, there is a phosphate lock loop (scene of loop) (residues 775–787) that connect the WEB and RuvC domains. Cas9 has four main mechanisms that are important for successful cleavage including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is a the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe and bridge helix interacts with the seed region of the <scene name='92/925538/Lobes_and_linkers/5'>sgRNA</scene> (C13-C20). The seed region is in the <scene name='92/925538/Lobes_and_linkers/8'>A-form conformation</scene> so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the <scene name='92/925538/Lobes_and_linkers/7'>phosphate backbone</scene>. The target DNA binds to the REC loop and RuvC domain for the proper conformation for base paring between the <scene name='92/925538/Lobes_and_linkers/9'>Target DNA and sgRNA</scene><ref name="Cas9" />.<br />
== Recognition of the PAM sequence ==<br />
For the recognition of the <scene name='92/925538/Lobes_and_linkers/10'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds (scene of bonds) as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.<br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the <scene name='92/925538/Lobes_and_linkers/11'>REC lobe and WED domain</scene>. The <scene name='92/925538/Lobes_and_linkers/11'>WED domain</scene> contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat:anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="Cas9" />.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, <scene name='92/925538/Lobes_and_linkers/12'>RuvC and HNH</scene> are in endonuclease activity (Scene). RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA <ref name="Cas9" />.<br />
</structuresection><br />
==References==<br />
<br />
<references /></div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3644021Sandbox Reserved 17502022-10-10T19:26:20Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single stranded guide RNA (sgRNA) to bind the target DNA that will be cut. Cas9 utalizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind to Cas9 to be cut. The PAM sequence acts as a two factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the REC lobe (residues 41–425) and NUC lobe (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WED, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The REC lobe is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site <ref>PMID:30555184</ref>. The RuvC nuclease domain (Scene of domain)(residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the [[sgRNA]] <ref name="sgRNA">PMID:24529477</ref>. The HNH domain (scene of domain) (residues 520–628) cleaves the target strand of DNA bound to the sgRNA <ref name="sgRNA" />. The WEB domain (Scene of domain) (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of twisted five-stranded beta sheet flanked by four alpha helices <ref>PMID:15596446</ref>. The PI domain (scene of domain) (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA <ref>PMID:24634220</ref>. There are also two linker domains (scene of domain), L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH <ref name="Cas9" />. Furthermore, there is a phosphate lock loop (scene of loop) (residues 775–787) that connect the WEB and RuvC domains. Cas9 has four main mechanisms that are important for successful cleavage including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is a the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe and bridge helix interacts with the seed region of the <scene name='92/925538/Lobes_and_linkers/5'>sgRNA</scene> (C13-C20). The seed region is in the <scene name='92/925538/Lobes_and_linkers/8'>A-form conformation</scene> so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the <scene name='92/925538/Lobes_and_linkers/7'>phosphate backbone</scene>. The target DNA binds to the REC loop and RuvC domain for the proper conformation for base paring between the <scene name='92/925538/Lobes_and_linkers/9'>Target DNA and sgRNA</scene><ref name="Cas9" />.<br />
== Recognition of the PAM sequence ==<br />
For the recognition of the <scene name='92/925538/Lobes_and_linkers/10'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds (scene of bonds) as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.<br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the <scene name='92/925538/Lobes_and_linkers/11'>REC lobe and WED domain</scene>. The <scene name='92/925538/Lobes_and_linkers/11'>WED domain</scene> contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat:anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="Cas9" />.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, <scene name='92/925538/Lobes_and_linkers/12'>RuvC and HNH</scene> are in endonuclease activity (Scene). RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA <ref name="Cas9" />.<br />
</structuresection><br />
==References==<br />
<br />
<references /></div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3644020Sandbox Reserved 17502022-10-10T19:03:26Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single stranded guide RNA (sgRNA) to bind the target DNA that will be cut. Cas9 utalizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind to Cas9 to be cut. The PAM sequence acts as a two factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the REC lobe (residues 41–425) and NUC lobe (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WED, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The REC lobe is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site <ref>PMID:30555184</ref>. The RuvC nuclease domain (Scene of domain)(residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the [[sgRNA]] <ref name="sgRNA">PMID:24529477</ref>. The HNH domain (scene of domain) (residues 520–628) cleaves the target strand of DNA bound to the sgRNA <ref name="sgRNA" />. The WEB domain (Scene of domain) (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of twisted five-stranded beta sheet flanked by four alpha helices <ref>PMID:15596446</ref>. The PI domain (scene of domain) (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA <ref>PMID:24634220</ref>. There are also two linker domains (scene of domain), L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH <ref name="Cas9" />. Furthermore, there is a phosphate lock loop (scene of loop) (residues 775–787) that connect the WEB and RuvC domains. Cas9 has four main mechanisms that are important for successful cleavage including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is a the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe and bridge helix interacts with the seed region of the <scene name='92/925538/Lobes_and_linkers/5'>sgRNA</scene> (C13-C20). The seed region is in the <scene name='92/925538/Lobes_and_linkers/8'>A-form conformation</scene> so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the <scene name='92/925538/Lobes_and_linkers/7'>phosphate backbone</scene>. The target DNA binds to the REC loop and RuvC domain for the proper conformation for base paring between the <scene name='92/925538/Lobes_and_linkers/9'>Target DNA and sgRNA</scene><ref name="Cas9" />.<br />
== Recognition of the PAM sequence ==<br />
For the recognition of the <scene name='92/925538/Lobes_and_linkers/10'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds (scene of bonds) as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.<br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the <scene name='92/925538/Lobes_and_linkers/11'>REC lobe and WED domain</scene>. The <scene name='92/925538/Lobes_and_linkers/11'>WED domain</scene> contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat:anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="Cas9" />.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, RuvC and HNH are in endonuclease activity (Scene). RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA <ref name="Cas9" />.<br />
</structuresection><br />
==References==<br />
<br />
<references /></div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3644017Sandbox Reserved 17502022-10-10T18:31:00Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single stranded guide RNA (sgRNA) to bind the target DNA that will be cut. Cas9 utalizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind to Cas9 to be cut. The PAM sequence acts as a two factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the REC lobe (residues 41–425) and NUC lobe (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WEB, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The REC lobe is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site <ref>PMID:30555184</ref>. The RuvC nuclease domain (Scene of domain)(residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the [[sgRNA]] <ref name="sgRNA">PMID:24529477</ref>. The HNH domain (scene of domain) (residues 520–628) cleaves the target strand of DNA bound to the sgRNA <ref name="sgRNA" />. The WEB domain (Scene of domain) (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of twisted five-stranded beta sheet flanked by four alpha helices <ref>PMID:15596446</ref>. The PI domain (scene of domain) (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA <ref>PMID:24634220</ref>. There are also two linker domains (scene of domain), L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH <ref name="Cas9" />. Furthermore, there is a phosphate lock loop (scene of loop) (residues 775–787) that connect the WEB and RuvC domains. Cas9 has four main mechanisms that are important for successful cleavage including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is a the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe and bridge helix interacts with the seed region of the <scene name='92/925538/Lobes_and_linkers/5'>sgRNA</scene> (C13-C20). The seed region is in the <scene name='92/925538/Lobes_and_linkers/8'>A-form conformation</scene> so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the <scene name='92/925538/Lobes_and_linkers/7'>phosphate backbone</scene>. The target DNA binds to the REC loop and RuvC domain for the proper conformation for base paring between the <scene name='92/925538/Lobes_and_linkers/9'>Target DNA and sgRNA</scene><ref name="Cas9" />.<br />
== Recognition of the PAM sequence ==<br />
For the recognition of the <scene name='92/925538/Lobes_and_linkers/10'>PAM sequence</scene>, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds (scene of bonds) as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.<br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the REC and WED domains (scene). The WED domain contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat:anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="Cas9" />.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, RuvC and HNH are in endonuclease activity (Scene). RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA <ref name="Cas9" />.<br />
</structuresection><br />
==References==<br />
<br />
<references /></div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3644007Sandbox Reserved 17502022-10-10T16:28:36Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single stranded guide RNA (sgRNA) to bind the target DNA that will be cut. Cas9 utalizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind to Cas9 to be cut. The PAM sequence acts as a two factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the REC lobe (residues 41–425) and NUC lobe (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WEB, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The REC lobe is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site <ref>PMID:30555184</ref>. The RuvC nuclease domain (Scene of domain)(residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the [[sgRNA]] <ref name="sgRNA">PMID:24529477</ref>. The HNH domain (scene of domain) (residues 520–628) cleaves the target strand of DNA bound to the sgRNA <ref name="sgRNA" />. The WEB domain (Scene of domain) (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of twisted five-stranded beta sheet flanked by four alpha helices <ref>PMID:15596446</ref>. The PI domain (scene of domain) (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA <ref>PMID:24634220</ref>. There are also two linker domains (scene of domain), L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH <ref name="Cas9" />. Furthermore, there is a phosphate lock loop (scene of loop) (residues 775–787) that connect the WEB and RuvC domains. Cas9 has four main mechanisms that are important for successful cleavage including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is a the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe and bridge helix interacts with the seed region of the <scene name='92/925538/Lobes_and_linkers/5'>sgRNA</scene> (C13-C20). The seed region is in the <scene name='92/925538/Lobes_and_linkers/8'>A-form conformation</scene> so it can bind the target DNA. Only the REC lobe interacts with the PAM distal region pf the sgRNA (A3-U6) through the <scene name='92/925538/Lobes_and_linkers/7'>phosphate backbone</scene>. The target DNA binds to the REC loop and RuvC domain for the proper conformation for base paring between the <scene name='92/925538/Lobes_and_linkers/9'>Target DNA and sgRNA</scene><ref name="Cas9" />.<br />
== Recognition of the PAM sequence ==<br />
For the recognition of the PAM sequence, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds (scene of bonds) as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.<br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the REC and WED domains (scene). The WED domain contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat:anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="Cas9" />.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, RuvC and HNH are in endonuclease activity (Scene). RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA <ref name="Cas9" />.<br />
</structuresection><br />
==References==<br />
<br />
<references /></div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3644006Sandbox Reserved 17502022-10-10T15:27:10Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single stranded guide RNA (sgRNA) to bind the target DNA that will be cut. Cas9 utalizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind to Cas9 to be cut. The PAM sequence acts as a two factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the REC lobe (residues 41–425) and NUC lobe (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WEB, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The REC lobe is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site <ref>PMID:30555184</ref>. The RuvC nuclease domain (Scene of domain)(residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the [[sgRNA]] <ref name="sgRNA">PMID:24529477</ref>. The HNH domain (scene of domain) (residues 520–628) cleaves the target strand of DNA bound to the sgRNA <ref name="sgRNA" />. The WEB domain (Scene of domain) (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of twisted five-stranded beta sheet flanked by four alpha helices <ref>PMID:15596446</ref>. The PI domain (scene of domain) (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA <ref>PMID:24634220</ref>. There are also two linker domains (scene of domain), L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH <ref name="Cas9" />. Furthermore, there is a phosphate lock loop (scene of loop) (residues 775–787) that connect the WEB and RuvC domains. Cas9 has four main mechanisms that are important for successful cleavage including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes (Scene). A heteroduplex is a the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe interacts with the seed region of the sgRNA (C13-C20) as well as the PAM distal region (A3-U6) through the phosphate backbone. The seed region is in the A-form confirmation so it can bind the target DNA. The target DNA binds to the REC loop and RuvC domain for the proper conformation for base paring between the target DNA and sgRNA <ref name="Cas9" />.<br />
== Recognition of the PAM sequence ==<br />
For the recognition of the PAM sequence, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds (scene of bonds) as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.<br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the REC and WED domains (scene). The WED domain contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat:anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="Cas9" />.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, RuvC and HNH are in endonuclease activity (Scene). RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA <ref name="Cas9" />.<br />
</structuresection><br />
==References==<br />
<br />
<references /></div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3644005Sandbox Reserved 17502022-10-10T15:23:28Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single stranded guide RNA (sgRNA) to bind the target DNA that will be cut. Cas9 utalizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind to Cas9 to be cut. The PAM sequence acts as a two factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the REC lobe (residues 41–425) and NUC lobe (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WEB, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The REC lobe is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site <ref>PMID:30555184</ref>. The RuvC nuclease domain (Scene of domain)(residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the sgRNA <ref>PMID:24529477</ref>. The HNH domain (scene of domain) (residues 520–628) cleaves the target strand of DNA bound to the sgRNA <ref>PMID:24529477</ref>. The WEB domain (Scene of domain) (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of twisted five-stranded beta sheet flanked by four alpha helices <ref>PMID:15596446</ref>. The PI domain (scene of domain) (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA <ref>PMID:24634220</ref>. There are also two linker domains (scene of domain), L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH <ref name="Cas9" />. Furthermore, there is a phosphate lock loop (scene of loop) (residues 775–787) that connect the WEB and RuvC domains. Cas9 has four main mechanisms that are important for successful cleavage including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes (Scene). A heteroduplex is a the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe interacts with the seed region of the sgRNA (C13-C20) as well as the PAM distal region (A3-U6) through the phosphate backbone. The seed region is in the A-form confirmation so it can bind the target DNA. The target DNA binds to the REC loop and RuvC domain for the proper conformation for base paring between the target DNA and sgRNA <ref name="Cas9" />.<br />
== Recognition of the PAM sequence ==<br />
For the recognition of the PAM sequence, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds (scene of bonds) as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="Cas9" />.<br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the REC and WED domains (scene). The WED domain contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat:anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="Cas9" />.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, RuvC and HNH are in endonuclease activity (Scene). RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA <ref name="Cas9" />.<br />
</structuresection><br />
==References==<br />
<br />
<references /></div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3644004Sandbox Reserved 17502022-10-10T15:22:49Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single stranded guide RNA (sgRNA) to bind the target DNA that will be cut. Cas9 utalizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind to Cas9 to be cut. The PAM sequence acts as a two factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the REC lobe (residues 41–425) and NUC lobe (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WEB, and PI domains in [[Cas9]] <ref name="Cas9">PMID:26317473</ref>. The REC lobe is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site <ref>PMID:30555184</ref>. The RuvC nuclease domain (Scene of domain)(residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the sgRNA <ref>PMID:24529477</ref>. The HNH domain (scene of domain) (residues 520–628) cleaves the target strand of DNA bound to the sgRNA <ref>PMID:24529477</ref>. The WEB domain (Scene of domain) (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of twisted five-stranded beta sheet flanked by four alpha helices <ref>PMID:15596446</ref>. The PI domain (scene of domain) (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA <ref>PMID:24634220</ref>. There are also two linker domains (scene of domain), L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH <ref name="SA Cas9" />. Furthermore, there is a phosphate lock loop (scene of loop) (residues 775–787) that connect the WEB and RuvC domains. Cas9 has four main mechanisms that are important for successful cleavage including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes (Scene). A heteroduplex is a the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe interacts with the seed region of the sgRNA (C13-C20) as well as the PAM distal region (A3-U6) through the phosphate backbone. The seed region is in the A-form confirmation so it can bind the target DNA. The target DNA binds to the REC loop and RuvC domain for the proper conformation for base paring between the target DNA and sgRNA <ref name="SA Cas9" />.<br />
== Recognition of the PAM sequence ==<br />
For the recognition of the PAM sequence, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds (scene of bonds) as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="SA Cas9" />.<br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the REC and WED domains (scene). The WED domain contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat:anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="SA Cas9" />.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, RuvC and HNH are in endonuclease activity (Scene). RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA <ref name="SA Cas9" />.<br />
</structuresection><br />
==References==<br />
<br />
<references /></div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3644003Sandbox Reserved 17502022-10-10T15:18:55Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single stranded guide RNA (sgRNA) to bind the target DNA that will be cut. Cas9 utalizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind to Cas9 to be cut. The PAM sequence acts as a two factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the REC lobe (residues 41–425) and NUC lobe (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WEB, and PI domains <refname="SA Cas9">PMID:26317473</ref>. The REC lobe is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site <ref>PMID:30555184</ref>. The RuvC nuclease domain (Scene of domain)(residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the sgRNA <ref>PMID:24529477</ref>. The HNH domain (scene of domain) (residues 520–628) cleaves the target strand of DNA bound to the sgRNA <ref>PMID:24529477</ref>. The WEB domain (Scene of domain) (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of twisted five-stranded beta sheet flanked by four alpha helices <ref>PMID:15596446</ref>. The PI domain (scene of domain) (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA <ref>PMID:24634220</ref>. There are also two linker domains (scene of domain), L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH <ref name="SA Cas9" />. Furthermore, there is a phosphate lock loop (scene of loop) (residues 775–787) that connect the WEB and RuvC domains. Cas9 has four main mechanisms that are important for successful cleavage including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes (Scene). A heteroduplex is a the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe interacts with the seed region of the sgRNA (C13-C20) as well as the PAM distal region (A3-U6) through the phosphate backbone. The seed region is in the A-form confirmation so it can bind the target DNA. The target DNA binds to the REC loop and RuvC domain for the proper conformation for base paring between the target DNA and sgRNA <ref name="SA Cas9" />.<br />
== Recognition of the PAM sequence ==<br />
For the recognition of the PAM sequence, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds (scene of bonds) as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex <ref name="SA Cas9" />.<br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the REC and WED domains (scene). The WED domain contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat:anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref name="SA Cas9" />.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, RuvC and HNH are in endonuclease activity (Scene). RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA <ref name="SA Cas9" />.<br />
</structuresection><br />
==References==<br />
<br />
<references /></div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3644002Sandbox Reserved 17502022-10-10T15:15:45Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single stranded guide RNA (sgRNA) to bind the target DNA that will be cut. Cas9 utalizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind to Cas9 to be cut. The PAM sequence acts as a two factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the REC lobe (residues 41–425) and NUC lobe (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WEB, and PI domains <ref>PMID:26317473</ref>. The REC lobe is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site <ref>PMID:30555184</ref>. The RuvC nuclease domain (Scene of domain)(residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the sgRNA <ref>PMID:24529477</ref>. The HNH domain (scene of domain) (residues 520–628) cleaves the target strand of DNA bound to the sgRNA <ref>PMID:24529477</ref>. The WEB domain (Scene of domain) (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of twisted five-stranded beta sheet flanked by four alpha helices <ref>PMID:15596446</ref>. The PI domain (scene of domain) (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA <ref>PMID:24634220</ref>. There are also two linker domains (scene of domain), L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH <ref>PMID:26317473</ref>. Furthermore, there is a phosphate lock loop (scene of loop) (residues 775–787) that connect the WEB and RuvC domains. Cas9 has four main mechanisms that are important for successful cleavage including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes (Scene). A heteroduplex is a the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe interacts with the seed region of the sgRNA (C13-C20) as well as the PAM distal region (A3-U6) through the phosphate backbone. The seed region is in the A-form confirmation so it can bind the target DNA. The target DNA binds to the REC loop and RuvC domain for the proper conformation for base paring between the target DNA and sgRNA <ref>PMID:26317473</ref>.<br />
== Recognition of the PAM sequence ==<br />
For the recognition of the PAM sequence, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds (scene of bonds) as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex <ref>PMID:26317473</ref>.<br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the REC and WED domains (scene). The WED domain contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat:anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9 <ref>PMID:26317473</ref>.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, RuvC and HNH are in endonuclease activity (Scene). RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA <ref>PMID:26317473</ref>.<br />
</structuresection><br />
==References==<br />
<br />
<references /></div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3644001Sandbox Reserved 17502022-10-10T15:07:42Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single stranded guide RNA (sgRNA) to bind the target DNA that will be cut. Cas9 utalizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind to Cas9 to be cut. The PAM sequence acts as a two factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the REC lobe (residues 41–425) and NUC lobe (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WEB, and PI domains <ref>PMID:26317473</ref>. The REC lobe is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site (2). The RuvC nuclease domain (Scene of domain)(residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the sgRNA (3). The HNH domain (scene of domain) (residues 520–628) cleaves the target strand of DNA bound to the sgRNA (3). The WEB domain (Scene of domain) (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of twisted five-stranded beta sheet flanked by four alpha helices (4). The PI domain (scene of domain) (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA (5). There are also two linker domains (scene of domain), L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH (1). Furthermore, there is a phosphate lock loop (scene of loop) (residues 775–787) that connect the WEB and RuvC domains. Cas9 has four main mechanisms that are important for successful cleavage including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes (Scene). A heteroduplex is a the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe interacts with the seed region of the sgRNA (C13-C20) as well as the PAM distal region (A3-U6) through the phosphate backbone. The seed region is in the A-form confirmation so it can bind the target DNA. The target DNA binds to the REC loop and RuvC domain for the proper conformation for base paring between the target DNA and sgRNA. <br />
== Recognition of the PAM sequence ==<br />
For the recognition of the PAM sequence, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds (scene of bonds) as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex. <br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the REC and WED domains (scene). The WED domain contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat:anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, RuvC and HNH are in endonuclease activity (Scene). RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA.<br />
</structuresection><br />
==References==<br />
<br />
<references /></div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3644000Sandbox Reserved 17502022-10-10T15:05:39Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single stranded guide RNA (sgRNA) to bind the target DNA that will be cut. Cas9 utalizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind to Cas9 to be cut. The PAM sequence acts as a two factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the REC lobe (residues 41–425) and NUC lobe (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WEB, and PI domains <ref>PMID:26317473</ref>. The REC lobe is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site (2). The RuvC nuclease domain (Scene of domain)(residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the sgRNA (3). The HNH domain (scene of domain) (residues 520–628) cleaves the target strand of DNA bound to the sgRNA (3). The WEB domain (Scene of domain) (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of twisted five-stranded beta sheet flanked by four alpha helices (4). The PI domain (scene of domain) (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA (5). There are also two linker domains (scene of domain), L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH (1). Furthermore, there is a phosphate lock loop (scene of loop) (residues 775–787) that connect the WEB and RuvC domains. Cas9 has four main mechanisms that are important for successful cleavage including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes (Scene). A heteroduplex is a the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe interacts with the seed region of the sgRNA (C13-C20) as well as the PAM distal region (A3-U6) through the phosphate backbone. The seed region is in the A-form confirmation so it can bind the target DNA. The target DNA binds to the REC loop and RuvC domain for the proper conformation for base paring between the target DNA and sgRNA. <br />
== Recognition of the PAM sequence ==<br />
For the recognition of the PAM sequence, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds (scene of bonds) as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex. <br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the REC and WED domains (scene). The WED domain contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat:anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, RuvC and HNH are in endonuclease activity (Scene). RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA.<br />
</structuresection><br />
== Citations ==<br />
1.<ref group="xtra">PMID:26317473</ref><br />
<br />
2. Palermo G, Chen JS, Ricci CG, Rivalta I, Jinek M, Batista VS, Doudna JA, McCammon JA. Key role of the REC lobe during CRISPR-Cas9 activation by 'sensing', 'regulating', and 'locking' the catalytic HNH domain. Q Rev Biophys. 2018;51:e91. doi: 10.1017/S0033583518000070. Epub 2018 Aug 3. PMID: 30555184; PMCID: PMC6292676.<br />
<br />
3. Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani R, Zhang F, Nureki O. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell. 2014 Feb 27;156(5):935-49. doi: 10.1016/j.cell.2014.02.001. Epub 2014 Feb 13. PMID: 24529477; PMCID: PMC4139937.<br />
<br />
4. Morlot C, Pernot L, Le Gouellec A, Di Guilmi AM, Vernet T, Dideberg O, Dessen A. Crystal structure of a peptidoglycan synthesis regulatory factor (PBP3) from Streptococcus pneumoniae. J Biol Chem. 2005 Apr 22;280(16):15984-91. doi: 10.1074/jbc.M408446200. Epub 2004 Dec 13. PMID: 15596446.<br />
<br />
5. Chen H, Choi J, Bailey S. Cut site selection by the two nuclease domains of the Cas9 RNA-guided endonuclease. J Biol Chem. 2014 May 9;289(19):13284-94. doi: 10.1074/jbc.M113.539726. Epub 2014 Mar 14. PMID: 24634220; PMCID: PMC4036338.</div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3643999Sandbox Reserved 17502022-10-10T15:04:22Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single stranded guide RNA (sgRNA) to bind the target DNA that will be cut. Cas9 utalizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind to Cas9 to be cut. The PAM sequence acts as a two factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the REC lobe (residues 41–425) and NUC lobe (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WEB, and PI domains <ref>PMID:26317473</ref>. The REC lobe is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site (2). The RuvC nuclease domain (Scene of domain)(residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the sgRNA (3). The HNH domain (scene of domain) (residues 520–628) cleaves the target strand of DNA bound to the sgRNA (3). The WEB domain (Scene of domain) (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of twisted five-stranded beta sheet flanked by four alpha helices (4). The PI domain (scene of domain) (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA (5). There are also two linker domains (scene of domain), L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH (1). Furthermore, there is a phosphate lock loop (scene of loop) (residues 775–787) that connect the WEB and RuvC domains. Cas9 has four main mechanisms that are important for successful cleavage including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes (Scene). A heteroduplex is a the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe interacts with the seed region of the sgRNA (C13-C20) as well as the PAM distal region (A3-U6) through the phosphate backbone. The seed region is in the A-form confirmation so it can bind the target DNA. The target DNA binds to the REC loop and RuvC domain for the proper conformation for base paring between the target DNA and sgRNA. <br />
== Recognition of the PAM sequence ==<br />
For the recognition of the PAM sequence, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds (scene of bonds) as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex. <br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the REC and WED domains (scene). The WED domain contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat:anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, RuvC and HNH are in endonuclease activity (Scene). RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA.<br />
</structuresection><br />
== Citations ==<br />
1. Hiroshi Nishimasu, Le Cong, Winston X. Yan, F. Ann Ran, Bernd Zetsche, Yinqing Li, Arisa Kurabayashi, Ryuichiro Ishitani, Feng Zhang, Osamu Nureki, Crystal Structure of Staphylococcus aureus Cas9, Cell,Volume 162, Issue 5,2015,Pages 1113-1126,ISSN 0092-8674,https://doi.org/10.1016/j.cell.2015.08.007.<br />
<br />
2. Palermo G, Chen JS, Ricci CG, Rivalta I, Jinek M, Batista VS, Doudna JA, McCammon JA. Key role of the REC lobe during CRISPR-Cas9 activation by 'sensing', 'regulating', and 'locking' the catalytic HNH domain. Q Rev Biophys. 2018;51:e91. doi: 10.1017/S0033583518000070. Epub 2018 Aug 3. PMID: 30555184; PMCID: PMC6292676.<br />
<br />
3. Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani R, Zhang F, Nureki O. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell. 2014 Feb 27;156(5):935-49. doi: 10.1016/j.cell.2014.02.001. Epub 2014 Feb 13. PMID: 24529477; PMCID: PMC4139937.<br />
<br />
4. Morlot C, Pernot L, Le Gouellec A, Di Guilmi AM, Vernet T, Dideberg O, Dessen A. Crystal structure of a peptidoglycan synthesis regulatory factor (PBP3) from Streptococcus pneumoniae. J Biol Chem. 2005 Apr 22;280(16):15984-91. doi: 10.1074/jbc.M408446200. Epub 2004 Dec 13. PMID: 15596446.<br />
<br />
5. Chen H, Choi J, Bailey S. Cut site selection by the two nuclease domains of the Cas9 RNA-guided endonuclease. J Biol Chem. 2014 May 9;289(19):13284-94. doi: 10.1074/jbc.M113.539726. Epub 2014 Mar 14. PMID: 24634220; PMCID: PMC4036338.</div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3643968Sandbox Reserved 17502022-10-09T17:02:42Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single stranded guide RNA (sgRNA) to bind the target DNA that will be cut. Cas9 utalizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind to Cas9 to be cut. The PAM sequence acts as a two factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. The main domains in the <scene name='92/925538/Lobes_and_linkers/4'>Cas9</scene> are the REC lobe (residues 41–425) and NUC lobe (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WEB, and PI domains (1). The REC lobe is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site (2). The RuvC nuclease domain (Scene of domain)(residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the sgRNA (3). The HNH domain (scene of domain) (residues 520–628) cleaves the target strand of DNA bound to the sgRNA (3). The WEB domain (Scene of domain) (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of twisted five-stranded beta sheet flanked by four alpha helices (4). The PI domain (scene of domain) (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA (5). There are also two linker domains (scene of domain), L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH (1). Furthermore, there is a phosphate lock loop (scene of loop) (residues 775–787) that connect the WEB and RuvC domains. Cas9 has four main mechanisms that are important for successful cleavage including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes (Scene). A heteroduplex is a the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe interacts with the seed region of the sgRNA (C13-C20) as well as the PAM distal region (A3-U6) through the phosphate backbone. The seed region is in the A-form confirmation so it can bind the target DNA. The target DNA binds to the REC loop and RuvC domain for the proper conformation for base paring between the target DNA and sgRNA. <br />
== Recognition of the PAM sequence ==<br />
For the recognition of the PAM sequence, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds (scene of bonds) as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex. <br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the REC and WED domains (scene). The WED domain contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat:anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, RuvC and HNH are in endonuclease activity (Scene). RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA.<br />
</structuresection><br />
== Citations ==<br />
1. Hiroshi Nishimasu, Le Cong, Winston X. Yan, F. Ann Ran, Bernd Zetsche, Yinqing Li, Arisa Kurabayashi, Ryuichiro Ishitani, Feng Zhang, Osamu Nureki, Crystal Structure of Staphylococcus aureus Cas9, Cell,Volume 162, Issue 5,2015,Pages 1113-1126,ISSN 0092-8674,https://doi.org/10.1016/j.cell.2015.08.007.<br />
<br />
2. Palermo G, Chen JS, Ricci CG, Rivalta I, Jinek M, Batista VS, Doudna JA, McCammon JA. Key role of the REC lobe during CRISPR-Cas9 activation by 'sensing', 'regulating', and 'locking' the catalytic HNH domain. Q Rev Biophys. 2018;51:e91. doi: 10.1017/S0033583518000070. Epub 2018 Aug 3. PMID: 30555184; PMCID: PMC6292676.<br />
<br />
3. Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani R, Zhang F, Nureki O. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell. 2014 Feb 27;156(5):935-49. doi: 10.1016/j.cell.2014.02.001. Epub 2014 Feb 13. PMID: 24529477; PMCID: PMC4139937.<br />
<br />
4. Morlot C, Pernot L, Le Gouellec A, Di Guilmi AM, Vernet T, Dideberg O, Dessen A. Crystal structure of a peptidoglycan synthesis regulatory factor (PBP3) from Streptococcus pneumoniae. J Biol Chem. 2005 Apr 22;280(16):15984-91. doi: 10.1074/jbc.M408446200. Epub 2004 Dec 13. PMID: 15596446.<br />
<br />
5. Chen H, Choi J, Bailey S. Cut site selection by the two nuclease domains of the Cas9 RNA-guided endonuclease. J Biol Chem. 2014 May 9;289(19):13284-94. doi: 10.1074/jbc.M113.539726. Epub 2014 Mar 14. PMID: 24634220; PMCID: PMC4036338.</div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3643967Sandbox Reserved 17502022-10-09T16:47:43Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA rendering it inert. <scene name='92/925538/Cas9_overview/4'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single stranded guide RNA (sgRNA) to bind the target DNA that will be cut. Cas9 utalizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind to Cas9 to be cut. The PAM sequence acts as a two factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. The main domains in the <scene name='92/925538/Lobes_and_linkers/3'>Cas9</scene> are the REC lobe (residues 41–425) and NUC lobe (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WEB, and PI domains (1). The REC lobe is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site (2). The RuvC nuclease domain (Scene of domain)(residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the sgRNA (3). The HNH domain (scene of domain) (residues 520–628) cleaves the target strand of DNA bound to the sgRNA (3). The WEB domain (Scene of domain) (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of twisted five-stranded beta sheet flanked by four alpha helices (4). The PI domain (scene of domain) (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA (5). There are also two linker domains (scene of domain), L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH (1). Furthermore, there is a phosphate lock loop (scene of loop) (residues 775–787) that connect the WEB and RuvC domains. Cas9 has four main mechanisms that are important for successful cleavage including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes (Scene). A heteroduplex is a the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe interacts with the seed region of the sgRNA (C13-C20) as well as the PAM distal region (A3-U6) through the phosphate backbone. The seed region is in the A-form confirmation so it can bind the target DNA. The target DNA binds to the REC loop and RuvC domain for the proper conformation for base paring between the target DNA and sgRNA. <br />
== Recognition of the PAM sequence ==<br />
For the recognition of the PAM sequence, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds (scene of bonds) as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex. <br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the REC and WED domains (scene). The WED domain contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat:anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, RuvC and HNH are in endonuclease activity (Scene). RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA.<br />
</structuresection><br />
== Citations ==<br />
1. Hiroshi Nishimasu, Le Cong, Winston X. Yan, F. Ann Ran, Bernd Zetsche, Yinqing Li, Arisa Kurabayashi, Ryuichiro Ishitani, Feng Zhang, Osamu Nureki, Crystal Structure of Staphylococcus aureus Cas9, Cell,Volume 162, Issue 5,2015,Pages 1113-1126,ISSN 0092-8674,https://doi.org/10.1016/j.cell.2015.08.007.<br />
<br />
2. Palermo G, Chen JS, Ricci CG, Rivalta I, Jinek M, Batista VS, Doudna JA, McCammon JA. Key role of the REC lobe during CRISPR-Cas9 activation by 'sensing', 'regulating', and 'locking' the catalytic HNH domain. Q Rev Biophys. 2018;51:e91. doi: 10.1017/S0033583518000070. Epub 2018 Aug 3. PMID: 30555184; PMCID: PMC6292676.<br />
<br />
3. Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani R, Zhang F, Nureki O. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell. 2014 Feb 27;156(5):935-49. doi: 10.1016/j.cell.2014.02.001. Epub 2014 Feb 13. PMID: 24529477; PMCID: PMC4139937.<br />
<br />
4. Morlot C, Pernot L, Le Gouellec A, Di Guilmi AM, Vernet T, Dideberg O, Dessen A. Crystal structure of a peptidoglycan synthesis regulatory factor (PBP3) from Streptococcus pneumoniae. J Biol Chem. 2005 Apr 22;280(16):15984-91. doi: 10.1074/jbc.M408446200. Epub 2004 Dec 13. PMID: 15596446.<br />
<br />
5. Chen H, Choi J, Bailey S. Cut site selection by the two nuclease domains of the Cas9 RNA-guided endonuclease. J Biol Chem. 2014 May 9;289(19):13284-94. doi: 10.1074/jbc.M113.539726. Epub 2014 Mar 14. PMID: 24634220; PMCID: PMC4036338.</div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3641362Sandbox Reserved 17502022-10-04T17:23:56Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA rendering it inert. <scene name='92/925538/Cas9_overview/2'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single stranded guide RNA (sgRNA) to bind the target DNA that will be cut. Cas9 utalizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind to Cas9 to be cut. The PAM sequence acts as a two factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. The main domains in the <scene name='92/925538/Lobes_and_linkers/3'>Cas9</scene> are the REC lobe (residues 41–425) and NUC lobe (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WEB, and PI domains (1). The REC lobe is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site (2). The RuvC nuclease domain (Scene of domain)(residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the sgRNA (3). The HNH domain (scene of domain) (residues 520–628) cleaves the target strand of DNA bound to the sgRNA (3). The WEB domain (Scene of domain) (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of twisted five-stranded beta sheet flanked by four alpha helices (4). The PI domain (scene of domain) (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA (5). There are also two linker domains (scene of domain), L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH (1). Furthermore, there is a phosphate lock loop (scene of loop) (residues 775–787) that connect the WEB and RuvC domains. Cas9 has four main mechanisms that are important for successful cleavage including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes (Scene). A heteroduplex is a the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe interacts with the seed region of the sgRNA (C13-C20) as well as the PAM distal region (A3-U6) through the phosphate backbone. The seed region is in the A-form confirmation so it can bind the target DNA. The target DNA binds to the REC loop and RuvC domain for the proper conformation for base paring between the target DNA and sgRNA. <br />
== Recognition of the PAM sequence ==<br />
For the recognition of the PAM sequence, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds (scene of bonds) as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex. <br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the REC and WED domains (scene). The WED domain contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat:anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, RuvC and HNH are in endonuclease activity (Scene). RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA.<br />
</structuresection><br />
== Citations ==<br />
1. Hiroshi Nishimasu, Le Cong, Winston X. Yan, F. Ann Ran, Bernd Zetsche, Yinqing Li, Arisa Kurabayashi, Ryuichiro Ishitani, Feng Zhang, Osamu Nureki, Crystal Structure of Staphylococcus aureus Cas9, Cell,Volume 162, Issue 5,2015,Pages 1113-1126,ISSN 0092-8674,https://doi.org/10.1016/j.cell.2015.08.007.<br />
<br />
2. Palermo G, Chen JS, Ricci CG, Rivalta I, Jinek M, Batista VS, Doudna JA, McCammon JA. Key role of the REC lobe during CRISPR-Cas9 activation by 'sensing', 'regulating', and 'locking' the catalytic HNH domain. Q Rev Biophys. 2018;51:e91. doi: 10.1017/S0033583518000070. Epub 2018 Aug 3. PMID: 30555184; PMCID: PMC6292676.<br />
<br />
3. Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani R, Zhang F, Nureki O. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell. 2014 Feb 27;156(5):935-49. doi: 10.1016/j.cell.2014.02.001. Epub 2014 Feb 13. PMID: 24529477; PMCID: PMC4139937.<br />
<br />
4. Morlot C, Pernot L, Le Gouellec A, Di Guilmi AM, Vernet T, Dideberg O, Dessen A. Crystal structure of a peptidoglycan synthesis regulatory factor (PBP3) from Streptococcus pneumoniae. J Biol Chem. 2005 Apr 22;280(16):15984-91. doi: 10.1074/jbc.M408446200. Epub 2004 Dec 13. PMID: 15596446.<br />
<br />
5. Chen H, Choi J, Bailey S. Cut site selection by the two nuclease domains of the Cas9 RNA-guided endonuclease. J Biol Chem. 2014 May 9;289(19):13284-94. doi: 10.1074/jbc.M113.539726. Epub 2014 Mar 14. PMID: 24634220; PMCID: PMC4036338.</div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3641361Sandbox Reserved 17502022-10-04T17:21:46Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA rendering it inert. <scene name='92/925538/Cas9_overview/2'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single stranded guide RNA (sgRNA) to bind the target DNA that will be cut. Cas9 utalizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind to Cas9 to be cut. The PAM sequence acts as a two factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. The main domains in the <scene name='92/925538/Lobes_and_linkers/3'>Cas9</scene> are the REC lobe (residues 41–425) and NUC lobe (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WEB, and PI domains (1). The REC lobe is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site (2). The RuvC nuclease domain (Scene of domain)(residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the sgRNA (3). The HNH domain (scene of domain) (residues 520–628) cleaves the target strand of DNA bound to the sgRNA (3). The WEB domain (Scene of domain) (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of twisted five-stranded beta sheet flanked by four alpha helices (4). The PI domain (scene of domain) (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA (5). There are also two linker domains (scene of domain), L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH (1). Furthermore, there is a phosphate lock loop (scene of loop) (residues 775–787) that connect the WEB and RuvC domains. Cas9 has four main mechanisms that are important for successful cleavage including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes (Scene). A heteroduplex is a the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe interacts with the seed region of the sgRNA (C13-C20) as well as the PAM distal region (A3-U6) through the phosphate backbone. The seed region is in the A-form confirmation so it can bind the target DNA. The target DNA binds to the REC loop and RuvC domain for the proper conformation for base paring between the target DNA and sgRNA. <br />
== Recognition of the PAM seequence ==<br />
For the recognition of the PAM sequence, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds (scene of bonds) as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex. <br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the REC and WED domains (scene). The WED domain contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat:anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, RuvC and HNH are in endonuclease activity (Scene). RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA.<br />
</structuresection><br />
== Citations ==<br />
1. Hiroshi Nishimasu, Le Cong, Winston X. Yan, F. Ann Ran, Bernd Zetsche, Yinqing Li, Arisa Kurabayashi, Ryuichiro Ishitani, Feng Zhang, Osamu Nureki, Crystal Structure of Staphylococcus aureus Cas9, Cell,Volume 162, Issue 5,2015,Pages 1113-1126,ISSN 0092-8674,https://doi.org/10.1016/j.cell.2015.08.007.<br />
<br />
2. Palermo G, Chen JS, Ricci CG, Rivalta I, Jinek M, Batista VS, Doudna JA, McCammon JA. Key role of the REC lobe during CRISPR-Cas9 activation by 'sensing', 'regulating', and 'locking' the catalytic HNH domain. Q Rev Biophys. 2018;51:e91. doi: 10.1017/S0033583518000070. Epub 2018 Aug 3. PMID: 30555184; PMCID: PMC6292676.<br />
<br />
3. Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani R, Zhang F, Nureki O. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell. 2014 Feb 27;156(5):935-49. doi: 10.1016/j.cell.2014.02.001. Epub 2014 Feb 13. PMID: 24529477; PMCID: PMC4139937.<br />
<br />
4. Morlot C, Pernot L, Le Gouellec A, Di Guilmi AM, Vernet T, Dideberg O, Dessen A. Crystal structure of a peptidoglycan synthesis regulatory factor (PBP3) from Streptococcus pneumoniae. J Biol Chem. 2005 Apr 22;280(16):15984-91. doi: 10.1074/jbc.M408446200. Epub 2004 Dec 13. PMID: 15596446.<br />
<br />
5. Chen H, Choi J, Bailey S. Cut site selection by the two nuclease domains of the Cas9 RNA-guided endonuclease. J Biol Chem. 2014 May 9;289(19):13284-94. doi: 10.1074/jbc.M113.539726. Epub 2014 Mar 14. PMID: 24634220; PMCID: PMC4036338.</div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3641360Sandbox Reserved 17502022-10-04T17:10:01Z<p>Thomas Gastineau: </p>
<hr />
<div>==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH sgRNA==<br />
<StructureSection load='5axw' size='340' side='right'caption='[[5axw]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA rendering it inert. <scene name='92/925538/Cas9_overview/2'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single stranded guide RNA (sgRNA) to bind the target DNA that will be cut. Cas9 utalizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind to Cas9 to be cut. The PAM sequence acts as a two factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. The main domains in the <scene name='92/925538/Lobes_and_linkers/2'>Cas9</scene> are the REC lobe (residues 41–425) and NUC lobe (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WEB, and PI domains (1). The REC lobe is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site (2). The RuvC nuclease domain (Scene of domain)(residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the sgRNA (3). The HNH domain (scene of domain) (residues 520–628) cleaves the target strand of DNA bound to the sgRNA (3). The WEB domain (Scene of domain) (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of twisted five-stranded beta sheet flanked by four alpha helices (4). The PI domain (scene of domain) (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA (5). There are also two linker domains (scene of domain), L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH (1). Furthermore, there is a phosphate lock loop (scene of loop) (residues 775–787) that connect the WEB and RuvC domains. Cas9 has four main mechanisms that are important for successful cleavage including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes (Scene). A heteroduplex is a the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe interacts with the seed region of the sgRNA (C13-C20) as well as the PAM distal region (A3-U6) through the phosphate backbone. The seed region is in the A-form confirmation so it can bind the target DNA. The target DNA binds to the REC loop and RuvC domain for the proper conformation for base paring between the target DNA and sgRNA. <br />
== Recognition of the PAM seequence ==<br />
For the recognition of the PAM sequence, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds (scene of bonds) as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex. <br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the REC and WED domains (scene). The WED domain contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat:anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, RuvC and HNH are in endonuclease activity (Scene). RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA.<br />
</structuresection><br />
== Citations ==<br />
1. Hiroshi Nishimasu, Le Cong, Winston X. Yan, F. Ann Ran, Bernd Zetsche, Yinqing Li, Arisa Kurabayashi, Ryuichiro Ishitani, Feng Zhang, Osamu Nureki, Crystal Structure of Staphylococcus aureus Cas9, Cell,Volume 162, Issue 5,2015,Pages 1113-1126,ISSN 0092-8674,https://doi.org/10.1016/j.cell.2015.08.007.<br />
<br />
2. Palermo G, Chen JS, Ricci CG, Rivalta I, Jinek M, Batista VS, Doudna JA, McCammon JA. Key role of the REC lobe during CRISPR-Cas9 activation by 'sensing', 'regulating', and 'locking' the catalytic HNH domain. Q Rev Biophys. 2018;51:e91. doi: 10.1017/S0033583518000070. Epub 2018 Aug 3. PMID: 30555184; PMCID: PMC6292676.<br />
<br />
3. Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani R, Zhang F, Nureki O. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell. 2014 Feb 27;156(5):935-49. doi: 10.1016/j.cell.2014.02.001. Epub 2014 Feb 13. PMID: 24529477; PMCID: PMC4139937.<br />
<br />
4. Morlot C, Pernot L, Le Gouellec A, Di Guilmi AM, Vernet T, Dideberg O, Dessen A. Crystal structure of a peptidoglycan synthesis regulatory factor (PBP3) from Streptococcus pneumoniae. J Biol Chem. 2005 Apr 22;280(16):15984-91. doi: 10.1074/jbc.M408446200. Epub 2004 Dec 13. PMID: 15596446.<br />
<br />
5. Chen H, Choi J, Bailey S. Cut site selection by the two nuclease domains of the Cas9 RNA-guided endonuclease. J Biol Chem. 2014 May 9;289(19):13284-94. doi: 10.1074/jbc.M113.539726. Epub 2014 Mar 14. PMID: 24634220; PMCID: PMC4036338.</div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3641359Sandbox Reserved 17502022-10-04T17:00:47Z<p>Thomas Gastineau: </p>
<hr />
<div>Displayed</scene></scene>==DNA RECOGNITION BY GAL4: STRUCTURE OF A PROTEIN/DNA COMPLEX==<br />
<StructureSection load='1d66' size='340' side='right'caption='[[1d66]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Structural highlights ==<br />
<table><tr><td colspan='2'>[[1d66]] is a 4 chain structure with sequence from [https://en.wikipedia.org/wiki/Atcc_18824 Atcc 18824]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1D66 OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=1D66 FirstGlance]. <br><br />
</td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=CD:CADMIUM+ION'>CD</scene></td></tr><br />
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=1d66 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1d66 OCA], [https://pdbe.org/1d66 PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=1d66 RCSB], [https://www.ebi.ac.uk/pdbsum/1d66 PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=1d66 ProSAT]</span></td></tr><br />
</table><br />
== Function ==<br />
[[https://www.uniprot.org/uniprot/GAL4_YEAST GAL4_YEAST]] This protein is a positive regulator for the gene expression of the galactose-induced genes such as GAL1, GAL2, GAL7, GAL10, and MEL1 which code for the enzymes used to convert galactose to glucose. It recognizes a 17 base pair sequence in (5'-CGGRNNRCYNYNCNCCG-3') the upstream activating sequence (UAS-G) of these genes. This sequence is correctly shown on the structure as (5'-CGGAGGACTGCCCTCCG-3'). <scene name='92/925538/Dna_protein_interaction/3'>The DNA-Protein Interaction</scene> with all of the base pairs within 5 angstroms of the protein highlighted illustrates that the protein interacts with both strands of the UAS.<br />
== Evolutionary Conservation ==<br />
[[Image:Consurf_key_small.gif|200px|right]]<br />
Check<jmol><br />
<jmolCheckbox><br />
<scriptWhenChecked>; select protein; define ~consurf_to_do selected; consurf_initial_scene = true; script "/wiki/ConSurf/d6/1d66_consurf.spt"</scriptWhenChecked><br />
<scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/initialview01.spt</scriptWhenUnchecked><br />
<text>to colour the structure by Evolutionary Conservation</text><br />
</jmolCheckbox><br />
</jmol>, as determined by [http://consurfdb.tau.ac.il/ ConSurfDB]. You may read the [[Conservation%2C_Evolutionary|explanation]] of the method and the full data available from [http://bental.tau.ac.il/new_ConSurfDB/main_output.php?pdb_ID=1d66 ConSurf].<br />
<div style="clear:both"></div><br />
<div style="background-color:#fffaf0;"><br />
== Publication Abstract from PubMed ==<br />
A specific DNA complex of the 65-residue, N-terminal fragment of the yeast transcriptional activator, GAL4, has been analysed at 2.7 A resolution by X-ray crystallography. The protein binds as a <scene name='92/925538/Dimer/2'>dimer</scene> to a symmetrical 17-base-pair sequence. There is a compact <scene name='92/925538/Metal_binding_domain/5'>metal binding domain</scene> (residues 8-40), an <scene name='92/925538/Extended_linker/4'>extended linker</scene> (41-49), and an <scene name='92/925538/Alpha-helical_dimerization/2'>alpha-helical dimerization element</scene> (50-64). A small, Zn(2+)-containing domain recognizes a conserved CCG triplet at each end of the site through direct contacts with the major groove. The metal binding domain contains <scene name='92/925538/Cysteine_metal_binding/3'>cysteine residues</scene> that coordinate to the metal as shown as cadmium. A short coiled-coil dimerization element imposes 2-fold symmetry. A segment of extended polypeptide chain links the metal-binding module to the dimerization element and specifies the length of the site. The relatively open structure of the complex would allow another protein to bind coordinately with GAL4.<br />
<br />
DNA recognition by GAL4: structure of a protein-DNA complex.,Marmorstein R, Carey M, Ptashne M, Harrison SC Nature. 1992 Apr 2;356(6368):408-14. PMID:1557122<ref>PMID:1557122</ref><br />
<br />
From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br><br />
</div><br />
<div class="pdbe-citations 1d66" style="background-color:#fffaf0;"></div><br />
<br />
==See Also==<br />
*[[Gal3-Gal80-Gal4|Gal3-Gal80-Gal4]]<br />
*[[Hydrogen in macromolecular models|Hydrogen in macromolecular models]]<br />
== References ==<br />
<references/><br />
__TOC__<br />
</StructureSection><br />
[[Category: Atcc 18824]]<br />
[[Category: Large Structures]]<br />
[[Category: Carey, M]]<br />
[[Category: Harrison, S C]]<br />
[[Category: Marmorstein, R]]<br />
[[Category: Ptashne, M]]<br />
[[Category: Double helix]]<br />
[[Category: Protein-dna complex]]<br />
[[Category: Transcription-dna complex]]<br />
<br />
==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH gRNA==<br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA rendering it inert. <scene name='92/925538/Cas9_overview/2'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single stranded guide RNA (sgRNA) to bind the target DNA that will be cut. Cas9 utalizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind to Cas9 to be cut. The PAM sequence acts as a two factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. The main domains in the <scene name='92/925538/Lobes_and_linkers/2'>Cas9</scene> are the REC lobe (residues 41–425) and NUC lobe (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WEB, and PI domains (1). The REC lobe is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site (2). The RuvC nuclease domain (Scene of domain)(residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the sgRNA (3). The HNH domain (scene of domain) (residues 520–628) cleaves the target strand of DNA bound to the sgRNA (3). The WEB domain (Scene of domain) (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of twisted five-stranded beta sheet flanked by four alpha helices (4). The PI domain (scene of domain) (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA (5). There are also two linker domains (scene of domain), L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH (1). Furthermore, there is a phosphate lock loop (scene of loop) (residues 775–787) that connect the WEB and RuvC domains. Cas9 has four main mechanisms that are important for successful cleavage including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes (Scene). A heteroduplex is a the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe interacts with the seed region of the sgRNA (C13-C20) as well as the PAM distal region (A3-U6) through the phosphate backbone. The seed region is in the A-form confirmation so it can bind the target DNA. The target DNA binds to the REC loop and RuvC domain for the proper conformation for base paring between the target DNA and sgRNA. <br />
== Recognition of the PAM seequence ==<br />
For the recognition of the PAM sequence, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds (scene of bonds) as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex. <br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the REC and WED domains (scene). The WED domain contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat:anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, RuvC and HNH are in endonuclease activity (Scene). RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA.<br />
<br />
== Citations ==<br />
1. Hiroshi Nishimasu, Le Cong, Winston X. Yan, F. Ann Ran, Bernd Zetsche, Yinqing Li, Arisa Kurabayashi, Ryuichiro Ishitani, Feng Zhang, Osamu Nureki, Crystal Structure of Staphylococcus aureus Cas9, Cell,Volume 162, Issue 5,2015,Pages 1113-1126,ISSN 0092-8674,https://doi.org/10.1016/j.cell.2015.08.007.<br />
<br />
2. Palermo G, Chen JS, Ricci CG, Rivalta I, Jinek M, Batista VS, Doudna JA, McCammon JA. Key role of the REC lobe during CRISPR-Cas9 activation by 'sensing', 'regulating', and 'locking' the catalytic HNH domain. Q Rev Biophys. 2018;51:e91. doi: 10.1017/S0033583518000070. Epub 2018 Aug 3. PMID: 30555184; PMCID: PMC6292676.<br />
<br />
3. Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani R, Zhang F, Nureki O. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell. 2014 Feb 27;156(5):935-49. doi: 10.1016/j.cell.2014.02.001. Epub 2014 Feb 13. PMID: 24529477; PMCID: PMC4139937.<br />
<br />
4. Morlot C, Pernot L, Le Gouellec A, Di Guilmi AM, Vernet T, Dideberg O, Dessen A. Crystal structure of a peptidoglycan synthesis regulatory factor (PBP3) from Streptococcus pneumoniae. J Biol Chem. 2005 Apr 22;280(16):15984-91. doi: 10.1074/jbc.M408446200. Epub 2004 Dec 13. PMID: 15596446.<br />
<br />
5. Chen H, Choi J, Bailey S. Cut site selection by the two nuclease domains of the Cas9 RNA-guided endonuclease. J Biol Chem. 2014 May 9;289(19):13284-94. doi: 10.1074/jbc.M113.539726. Epub 2014 Mar 14. PMID: 24634220; PMCID: PMC4036338.</div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3641357Sandbox Reserved 17502022-10-04T16:52:36Z<p>Thomas Gastineau: </p>
<hr />
<div>Displayed</scene></scene>==DNA RECOGNITION BY GAL4: STRUCTURE OF A PROTEIN/DNA COMPLEX==<br />
<StructureSection load='1d66' size='340' side='right'caption='[[1d66]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Structural highlights ==<br />
<table><tr><td colspan='2'>[[1d66]] is a 4 chain structure with sequence from [https://en.wikipedia.org/wiki/Atcc_18824 Atcc 18824]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1D66 OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=1D66 FirstGlance]. <br><br />
</td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=CD:CADMIUM+ION'>CD</scene></td></tr><br />
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=1d66 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1d66 OCA], [https://pdbe.org/1d66 PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=1d66 RCSB], [https://www.ebi.ac.uk/pdbsum/1d66 PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=1d66 ProSAT]</span></td></tr><br />
</table><br />
== Function ==<br />
[[https://www.uniprot.org/uniprot/GAL4_YEAST GAL4_YEAST]] This protein is a positive regulator for the gene expression of the galactose-induced genes such as GAL1, GAL2, GAL7, GAL10, and MEL1 which code for the enzymes used to convert galactose to glucose. It recognizes a 17 base pair sequence in (5'-CGGRNNRCYNYNCNCCG-3') the upstream activating sequence (UAS-G) of these genes. This sequence is correctly shown on the structure as (5'-CGGAGGACTGCCCTCCG-3'). <scene name='92/925538/Dna_protein_interaction/3'>The DNA-Protein Interaction</scene> with all of the base pairs within 5 angstroms of the protein highlighted illustrates that the protein interacts with both strands of the UAS.<br />
== Evolutionary Conservation ==<br />
[[Image:Consurf_key_small.gif|200px|right]]<br />
Check<jmol><br />
<jmolCheckbox><br />
<scriptWhenChecked>; select protein; define ~consurf_to_do selected; consurf_initial_scene = true; script "/wiki/ConSurf/d6/1d66_consurf.spt"</scriptWhenChecked><br />
<scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/initialview01.spt</scriptWhenUnchecked><br />
<text>to colour the structure by Evolutionary Conservation</text><br />
</jmolCheckbox><br />
</jmol>, as determined by [http://consurfdb.tau.ac.il/ ConSurfDB]. You may read the [[Conservation%2C_Evolutionary|explanation]] of the method and the full data available from [http://bental.tau.ac.il/new_ConSurfDB/main_output.php?pdb_ID=1d66 ConSurf].<br />
<div style="clear:both"></div><br />
<div style="background-color:#fffaf0;"><br />
== Publication Abstract from PubMed ==<br />
A specific DNA complex of the 65-residue, N-terminal fragment of the yeast transcriptional activator, GAL4, has been analysed at 2.7 A resolution by X-ray crystallography. The protein binds as a <scene name='92/925538/Dimer/2'>dimer</scene> to a symmetrical 17-base-pair sequence. There is a compact <scene name='92/925538/Metal_binding_domain/5'>metal binding domain</scene> (residues 8-40), an <scene name='92/925538/Extended_linker/4'>extended linker</scene> (41-49), and an <scene name='92/925538/Alpha-helical_dimerization/2'>alpha-helical dimerization element</scene> (50-64). A small, Zn(2+)-containing domain recognizes a conserved CCG triplet at each end of the site through direct contacts with the major groove. The metal binding domain contains <scene name='92/925538/Cysteine_metal_binding/3'>cysteine residues</scene> that coordinate to the metal as shown as cadmium. A short coiled-coil dimerization element imposes 2-fold symmetry. A segment of extended polypeptide chain links the metal-binding module to the dimerization element and specifies the length of the site. The relatively open structure of the complex would allow another protein to bind coordinately with GAL4.<br />
<br />
DNA recognition by GAL4: structure of a protein-DNA complex.,Marmorstein R, Carey M, Ptashne M, Harrison SC Nature. 1992 Apr 2;356(6368):408-14. PMID:1557122<ref>PMID:1557122</ref><br />
<br />
From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br><br />
</div><br />
<div class="pdbe-citations 1d66" style="background-color:#fffaf0;"></div><br />
<br />
==See Also==<br />
*[[Gal3-Gal80-Gal4|Gal3-Gal80-Gal4]]<br />
*[[Hydrogen in macromolecular models|Hydrogen in macromolecular models]]<br />
== References ==<br />
<references/><br />
__TOC__<br />
</StructureSection><br />
[[Category: Atcc 18824]]<br />
[[Category: Large Structures]]<br />
[[Category: Carey, M]]<br />
[[Category: Harrison, S C]]<br />
[[Category: Marmorstein, R]]<br />
[[Category: Ptashne, M]]<br />
[[Category: Double helix]]<br />
[[Category: Protein-dna complex]]<br />
[[Category: Transcription-dna complex]]<br />
<br />
==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH gRNA==<br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA rendering it inert. <scene name='92/925538/Cas9_overview/2'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single stranded guide RNA (sgRNA) to bind the target DNA that will be cut. Cas9 utalizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind to Cas9 to be cut. The PAM sequence acts as a two factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. The main domains in the <scene name='92/925538/Lobes_and_linkers/2'>Cas9</scene> are the REC lobe (residues 41–425) and NUC lobe (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WEB, and PI domains (1). The REC lobe is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site (2). The RuvC nuclease domain(residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the sgRNA (3). The HNH domain (residues 520–628) cleaves the target strand of DNA bound to the sgRNA (3). The WEB domain (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of twisted five-stranded beta sheet flanked by four alpha helices (4). The PI domain (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA (5). There are also two linker domains, L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH (1). Furthermore, there is a phosphate lock loop (residues 775–787) that connect the WEB and RuvC domains. Cas9 has four main mechanisms that are important for successful cleavage including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is a the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe interacts with the seed region of the sgRNA (C13-C20) as well as the PAM distal region (A3-U6) through the phosphate backbone. The seed region is in the A-form confirmation so it can bind the target DNA. The target DNA binds to the REC loop and RuvC domain for the proper conformation for base paring between the target DNA and sgRNA. <br />
== Recognition of the PAM seequence ==<br />
For the recognition of the PAM sequence, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex. <br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the REC and WED domains. The WED domain contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat:anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, RuvC and HNH are in endonuclease activity. RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA.<br />
<br />
== Citations ==<br />
1. Hiroshi Nishimasu, Le Cong, Winston X. Yan, F. Ann Ran, Bernd Zetsche, Yinqing Li, Arisa Kurabayashi, Ryuichiro Ishitani, Feng Zhang, Osamu Nureki, Crystal Structure of Staphylococcus aureus Cas9, Cell,Volume 162, Issue 5,2015,Pages 1113-1126,ISSN 0092-8674,https://doi.org/10.1016/j.cell.2015.08.007.<br />
<br />
2. Palermo G, Chen JS, Ricci CG, Rivalta I, Jinek M, Batista VS, Doudna JA, McCammon JA. Key role of the REC lobe during CRISPR-Cas9 activation by 'sensing', 'regulating', and 'locking' the catalytic HNH domain. Q Rev Biophys. 2018;51:e91. doi: 10.1017/S0033583518000070. Epub 2018 Aug 3. PMID: 30555184; PMCID: PMC6292676.<br />
<br />
3. Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani R, Zhang F, Nureki O. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell. 2014 Feb 27;156(5):935-49. doi: 10.1016/j.cell.2014.02.001. Epub 2014 Feb 13. PMID: 24529477; PMCID: PMC4139937.<br />
<br />
4. Morlot C, Pernot L, Le Gouellec A, Di Guilmi AM, Vernet T, Dideberg O, Dessen A. Crystal structure of a peptidoglycan synthesis regulatory factor (PBP3) from Streptococcus pneumoniae. J Biol Chem. 2005 Apr 22;280(16):15984-91. doi: 10.1074/jbc.M408446200. Epub 2004 Dec 13. PMID: 15596446.<br />
<br />
5. Chen H, Choi J, Bailey S. Cut site selection by the two nuclease domains of the Cas9 RNA-guided endonuclease. J Biol Chem. 2014 May 9;289(19):13284-94. doi: 10.1074/jbc.M113.539726. Epub 2014 Mar 14. PMID: 24634220; PMCID: PMC4036338.</div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3641356Sandbox Reserved 17502022-10-04T16:43:55Z<p>Thomas Gastineau: </p>
<hr />
<div>Displayed</scene></scene>==DNA RECOGNITION BY GAL4: STRUCTURE OF A PROTEIN/DNA COMPLEX==<br />
<StructureSection load='1d66' size='340' side='right'caption='[[1d66]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Structural highlights ==<br />
<table><tr><td colspan='2'>[[1d66]] is a 4 chain structure with sequence from [https://en.wikipedia.org/wiki/Atcc_18824 Atcc 18824]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1D66 OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=1D66 FirstGlance]. <br><br />
</td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=CD:CADMIUM+ION'>CD</scene></td></tr><br />
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=1d66 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1d66 OCA], [https://pdbe.org/1d66 PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=1d66 RCSB], [https://www.ebi.ac.uk/pdbsum/1d66 PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=1d66 ProSAT]</span></td></tr><br />
</table><br />
== Function ==<br />
[[https://www.uniprot.org/uniprot/GAL4_YEAST GAL4_YEAST]] This protein is a positive regulator for the gene expression of the galactose-induced genes such as GAL1, GAL2, GAL7, GAL10, and MEL1 which code for the enzymes used to convert galactose to glucose. It recognizes a 17 base pair sequence in (5'-CGGRNNRCYNYNCNCCG-3') the upstream activating sequence (UAS-G) of these genes. This sequence is correctly shown on the structure as (5'-CGGAGGACTGCCCTCCG-3'). <scene name='92/925538/Dna_protein_interaction/3'>The DNA-Protein Interaction</scene> with all of the base pairs within 5 angstroms of the protein highlighted illustrates that the protein interacts with both strands of the UAS.<br />
== Evolutionary Conservation ==<br />
[[Image:Consurf_key_small.gif|200px|right]]<br />
Check<jmol><br />
<jmolCheckbox><br />
<scriptWhenChecked>; select protein; define ~consurf_to_do selected; consurf_initial_scene = true; script "/wiki/ConSurf/d6/1d66_consurf.spt"</scriptWhenChecked><br />
<scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/initialview01.spt</scriptWhenUnchecked><br />
<text>to colour the structure by Evolutionary Conservation</text><br />
</jmolCheckbox><br />
</jmol>, as determined by [http://consurfdb.tau.ac.il/ ConSurfDB]. You may read the [[Conservation%2C_Evolutionary|explanation]] of the method and the full data available from [http://bental.tau.ac.il/new_ConSurfDB/main_output.php?pdb_ID=1d66 ConSurf].<br />
<div style="clear:both"></div><br />
<div style="background-color:#fffaf0;"><br />
== Publication Abstract from PubMed ==<br />
A specific DNA complex of the 65-residue, N-terminal fragment of the yeast transcriptional activator, GAL4, has been analysed at 2.7 A resolution by X-ray crystallography. The protein binds as a <scene name='92/925538/Dimer/2'>dimer</scene> to a symmetrical 17-base-pair sequence. There is a compact <scene name='92/925538/Metal_binding_domain/5'>metal binding domain</scene> (residues 8-40), an <scene name='92/925538/Extended_linker/4'>extended linker</scene> (41-49), and an <scene name='92/925538/Alpha-helical_dimerization/2'>alpha-helical dimerization element</scene> (50-64). A small, Zn(2+)-containing domain recognizes a conserved CCG triplet at each end of the site through direct contacts with the major groove. The metal binding domain contains <scene name='92/925538/Cysteine_metal_binding/3'>cysteine residues</scene> that coordinate to the metal as shown as cadmium. A short coiled-coil dimerization element imposes 2-fold symmetry. A segment of extended polypeptide chain links the metal-binding module to the dimerization element and specifies the length of the site. The relatively open structure of the complex would allow another protein to bind coordinately with GAL4.<br />
<br />
DNA recognition by GAL4: structure of a protein-DNA complex.,Marmorstein R, Carey M, Ptashne M, Harrison SC Nature. 1992 Apr 2;356(6368):408-14. PMID:1557122<ref>PMID:1557122</ref><br />
<br />
From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br><br />
</div><br />
<div class="pdbe-citations 1d66" style="background-color:#fffaf0;"></div><br />
<br />
==See Also==<br />
*[[Gal3-Gal80-Gal4|Gal3-Gal80-Gal4]]<br />
*[[Hydrogen in macromolecular models|Hydrogen in macromolecular models]]<br />
== References ==<br />
<references/><br />
__TOC__<br />
</StructureSection><br />
[[Category: Atcc 18824]]<br />
[[Category: Large Structures]]<br />
[[Category: Carey, M]]<br />
[[Category: Harrison, S C]]<br />
[[Category: Marmorstein, R]]<br />
[[Category: Ptashne, M]]<br />
[[Category: Double helix]]<br />
[[Category: Protein-dna complex]]<br />
[[Category: Transcription-dna complex]]<br />
<br />
==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH gRNA==<br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA rendering it inert. <scene name='92/925538/Cas9_overview/1'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single stranded guide RNA (sgRNA) to bind the target DNA that will be cut. Cas9 utalizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind to Cas9 to be cut. The PAM sequence acts as a two factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. The main domains in the <scene name='92/925538/Lobes_and_linkers/2'>Cas9</scene> are the REC lobe (residues 41–425) and NUC lobe (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WEB, and PI domains (1). The REC lobe is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site (2). The RuvC nuclease domain(residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the sgRNA (3). The HNH domain (residues 520–628) cleaves the target strand of DNA bound to the sgRNA (3). The WEB domain (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of twisted five-stranded beta sheet flanked by four alpha helices (4). The PI domain (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA (5). There are also two linker domains, L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH (1). Furthermore, there is a phosphate lock loop (residues 775–787) that connect the WEB and RuvC domains. Cas9 has four main mechanisms that are important for successful cleavage including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is a the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe interacts with the seed region of the sgRNA (C13-C20) as well as the PAM distal region (A3-U6) through the phosphate backbone. The seed region is in the A-form confirmation so it can bind the target DNA. The target DNA binds to the REC loop and RuvC domain for the proper conformation for base paring between the target DNA and sgRNA. <br />
== Recognition of the PAM seequence ==<br />
For the recognition of the PAM sequence, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex. <br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the REC and WED domains. The WED domain contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat:anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, RuvC and HNH are in endonuclease activity. RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA.<br />
<br />
== Citations ==<br />
1. Hiroshi Nishimasu, Le Cong, Winston X. Yan, F. Ann Ran, Bernd Zetsche, Yinqing Li, Arisa Kurabayashi, Ryuichiro Ishitani, Feng Zhang, Osamu Nureki, Crystal Structure of Staphylococcus aureus Cas9, Cell,Volume 162, Issue 5,2015,Pages 1113-1126,ISSN 0092-8674,https://doi.org/10.1016/j.cell.2015.08.007.<br />
<br />
2. Palermo G, Chen JS, Ricci CG, Rivalta I, Jinek M, Batista VS, Doudna JA, McCammon JA. Key role of the REC lobe during CRISPR-Cas9 activation by 'sensing', 'regulating', and 'locking' the catalytic HNH domain. Q Rev Biophys. 2018;51:e91. doi: 10.1017/S0033583518000070. Epub 2018 Aug 3. PMID: 30555184; PMCID: PMC6292676.<br />
<br />
3. Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani R, Zhang F, Nureki O. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell. 2014 Feb 27;156(5):935-49. doi: 10.1016/j.cell.2014.02.001. Epub 2014 Feb 13. PMID: 24529477; PMCID: PMC4139937.<br />
<br />
4. Morlot C, Pernot L, Le Gouellec A, Di Guilmi AM, Vernet T, Dideberg O, Dessen A. Crystal structure of a peptidoglycan synthesis regulatory factor (PBP3) from Streptococcus pneumoniae. J Biol Chem. 2005 Apr 22;280(16):15984-91. doi: 10.1074/jbc.M408446200. Epub 2004 Dec 13. PMID: 15596446.<br />
<br />
5. Chen H, Choi J, Bailey S. Cut site selection by the two nuclease domains of the Cas9 RNA-guided endonuclease. J Biol Chem. 2014 May 9;289(19):13284-94. doi: 10.1074/jbc.M113.539726. Epub 2014 Mar 14. PMID: 24634220; PMCID: PMC4036338.</div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3641355Sandbox Reserved 17502022-10-04T16:37:00Z<p>Thomas Gastineau: </p>
<hr />
<div>Displayed</scene></scene>==DNA RECOGNITION BY GAL4: STRUCTURE OF A PROTEIN/DNA COMPLEX==<br />
<StructureSection load='1d66' size='340' side='right'caption='[[1d66]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Structural highlights ==<br />
<table><tr><td colspan='2'>[[1d66]] is a 4 chain structure with sequence from [https://en.wikipedia.org/wiki/Atcc_18824 Atcc 18824]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1D66 OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=1D66 FirstGlance]. <br><br />
</td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=CD:CADMIUM+ION'>CD</scene></td></tr><br />
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=1d66 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1d66 OCA], [https://pdbe.org/1d66 PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=1d66 RCSB], [https://www.ebi.ac.uk/pdbsum/1d66 PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=1d66 ProSAT]</span></td></tr><br />
</table><br />
== Function ==<br />
[[https://www.uniprot.org/uniprot/GAL4_YEAST GAL4_YEAST]] This protein is a positive regulator for the gene expression of the galactose-induced genes such as GAL1, GAL2, GAL7, GAL10, and MEL1 which code for the enzymes used to convert galactose to glucose. It recognizes a 17 base pair sequence in (5'-CGGRNNRCYNYNCNCCG-3') the upstream activating sequence (UAS-G) of these genes. This sequence is correctly shown on the structure as (5'-CGGAGGACTGCCCTCCG-3'). <scene name='92/925538/Dna_protein_interaction/3'>The DNA-Protein Interaction</scene> with all of the base pairs within 5 angstroms of the protein highlighted illustrates that the protein interacts with both strands of the UAS.<br />
== Evolutionary Conservation ==<br />
[[Image:Consurf_key_small.gif|200px|right]]<br />
Check<jmol><br />
<jmolCheckbox><br />
<scriptWhenChecked>; select protein; define ~consurf_to_do selected; consurf_initial_scene = true; script "/wiki/ConSurf/d6/1d66_consurf.spt"</scriptWhenChecked><br />
<scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/initialview01.spt</scriptWhenUnchecked><br />
<text>to colour the structure by Evolutionary Conservation</text><br />
</jmolCheckbox><br />
</jmol>, as determined by [http://consurfdb.tau.ac.il/ ConSurfDB]. You may read the [[Conservation%2C_Evolutionary|explanation]] of the method and the full data available from [http://bental.tau.ac.il/new_ConSurfDB/main_output.php?pdb_ID=1d66 ConSurf].<br />
<div style="clear:both"></div><br />
<div style="background-color:#fffaf0;"><br />
== Publication Abstract from PubMed ==<br />
A specific DNA complex of the 65-residue, N-terminal fragment of the yeast transcriptional activator, GAL4, has been analysed at 2.7 A resolution by X-ray crystallography. The protein binds as a <scene name='92/925538/Dimer/2'>dimer</scene> to a symmetrical 17-base-pair sequence. There is a compact <scene name='92/925538/Metal_binding_domain/5'>metal binding domain</scene> (residues 8-40), an <scene name='92/925538/Extended_linker/4'>extended linker</scene> (41-49), and an <scene name='92/925538/Alpha-helical_dimerization/2'>alpha-helical dimerization element</scene> (50-64). A small, Zn(2+)-containing domain recognizes a conserved CCG triplet at each end of the site through direct contacts with the major groove. The metal binding domain contains <scene name='92/925538/Cysteine_metal_binding/3'>cysteine residues</scene> that coordinate to the metal as shown as cadmium. A short coiled-coil dimerization element imposes 2-fold symmetry. A segment of extended polypeptide chain links the metal-binding module to the dimerization element and specifies the length of the site. The relatively open structure of the complex would allow another protein to bind coordinately with GAL4.<br />
<br />
DNA recognition by GAL4: structure of a protein-DNA complex.,Marmorstein R, Carey M, Ptashne M, Harrison SC Nature. 1992 Apr 2;356(6368):408-14. PMID:1557122<ref>PMID:1557122</ref><br />
<br />
From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br><br />
</div><br />
<div class="pdbe-citations 1d66" style="background-color:#fffaf0;"></div><br />
<br />
==See Also==<br />
*[[Gal3-Gal80-Gal4|Gal3-Gal80-Gal4]]<br />
*[[Hydrogen in macromolecular models|Hydrogen in macromolecular models]]<br />
== References ==<br />
<references/><br />
__TOC__<br />
</StructureSection><br />
[[Category: Atcc 18824]]<br />
[[Category: Large Structures]]<br />
[[Category: Carey, M]]<br />
[[Category: Harrison, S C]]<br />
[[Category: Marmorstein, R]]<br />
[[Category: Ptashne, M]]<br />
[[Category: Double helix]]<br />
[[Category: Protein-dna complex]]<br />
[[Category: Transcription-dna complex]]<br />
<br />
==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH gRNA==<br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA rendering it inert. <scene name='92/925538/4un3/3'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single stranded guide RNA (sgRNA) to bind the target DNA that will be cut. Cas9 utalizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind to Cas9 to be cut. The PAM sequence acts as a two factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. The main domains in the <scene name='92/925538/Lobes_and_linkers/2'>Cas9</scene> are the REC lobe (residues 41–425) and NUC lobe (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WEB, and PI domains (1). The REC lobe is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site (2). The RuvC nuclease domain(residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the sgRNA (3). The HNH domain (residues 520–628) cleaves the target strand of DNA bound to the sgRNA (3). The WEB domain (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of twisted five-stranded beta sheet flanked by four alpha helices (4). The PI domain (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA (5). There are also two linker domains, L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH (1). Furthermore, there is a phosphate lock loop (residues 775–787) that connect the WEB and RuvC domains. Cas9 has four main mechanisms that are important for successful cleavage including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is a the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe interacts with the seed region of the sgRNA (C13-C20) as well as the PAM distal region (A3-U6) through the phosphate backbone. The seed region is in the A-form confirmation so it can bind the target DNA. The target DNA binds to the REC loop and RuvC domain for the proper conformation for base paring between the target DNA and sgRNA. <br />
== Recognition of the PAM seequence ==<br />
For the recognition of the PAM sequence, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex. <br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the REC and WED domains. The WED domain contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat:anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, RuvC and HNH are in endonuclease activity. RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA.<br />
<br />
== Citations ==<br />
1. Hiroshi Nishimasu, Le Cong, Winston X. Yan, F. Ann Ran, Bernd Zetsche, Yinqing Li, Arisa Kurabayashi, Ryuichiro Ishitani, Feng Zhang, Osamu Nureki, Crystal Structure of Staphylococcus aureus Cas9, Cell,Volume 162, Issue 5,2015,Pages 1113-1126,ISSN 0092-8674,https://doi.org/10.1016/j.cell.2015.08.007.<br />
<br />
2. Palermo G, Chen JS, Ricci CG, Rivalta I, Jinek M, Batista VS, Doudna JA, McCammon JA. Key role of the REC lobe during CRISPR-Cas9 activation by 'sensing', 'regulating', and 'locking' the catalytic HNH domain. Q Rev Biophys. 2018;51:e91. doi: 10.1017/S0033583518000070. Epub 2018 Aug 3. PMID: 30555184; PMCID: PMC6292676.<br />
<br />
3. Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani R, Zhang F, Nureki O. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell. 2014 Feb 27;156(5):935-49. doi: 10.1016/j.cell.2014.02.001. Epub 2014 Feb 13. PMID: 24529477; PMCID: PMC4139937.<br />
<br />
4. Morlot C, Pernot L, Le Gouellec A, Di Guilmi AM, Vernet T, Dideberg O, Dessen A. Crystal structure of a peptidoglycan synthesis regulatory factor (PBP3) from Streptococcus pneumoniae. J Biol Chem. 2005 Apr 22;280(16):15984-91. doi: 10.1074/jbc.M408446200. Epub 2004 Dec 13. PMID: 15596446.<br />
<br />
5. Chen H, Choi J, Bailey S. Cut site selection by the two nuclease domains of the Cas9 RNA-guided endonuclease. J Biol Chem. 2014 May 9;289(19):13284-94. doi: 10.1074/jbc.M113.539726. Epub 2014 Mar 14. PMID: 24634220; PMCID: PMC4036338.</div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3641349Sandbox Reserved 17502022-10-04T15:03:33Z<p>Thomas Gastineau: </p>
<hr />
<div>Displayed</scene></scene>==DNA RECOGNITION BY GAL4: STRUCTURE OF A PROTEIN/DNA COMPLEX==<br />
<StructureSection load='1d66' size='340' side='right'caption='[[1d66]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Structural highlights ==<br />
<table><tr><td colspan='2'>[[1d66]] is a 4 chain structure with sequence from [https://en.wikipedia.org/wiki/Atcc_18824 Atcc 18824]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1D66 OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=1D66 FirstGlance]. <br><br />
</td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=CD:CADMIUM+ION'>CD</scene></td></tr><br />
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=1d66 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1d66 OCA], [https://pdbe.org/1d66 PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=1d66 RCSB], [https://www.ebi.ac.uk/pdbsum/1d66 PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=1d66 ProSAT]</span></td></tr><br />
</table><br />
== Function ==<br />
[[https://www.uniprot.org/uniprot/GAL4_YEAST GAL4_YEAST]] This protein is a positive regulator for the gene expression of the galactose-induced genes such as GAL1, GAL2, GAL7, GAL10, and MEL1 which code for the enzymes used to convert galactose to glucose. It recognizes a 17 base pair sequence in (5'-CGGRNNRCYNYNCNCCG-3') the upstream activating sequence (UAS-G) of these genes. This sequence is correctly shown on the structure as (5'-CGGAGGACTGCCCTCCG-3'). <scene name='92/925538/Dna_protein_interaction/3'>The DNA-Protein Interaction</scene> with all of the base pairs within 5 angstroms of the protein highlighted illustrates that the protein interacts with both strands of the UAS.<br />
== Evolutionary Conservation ==<br />
[[Image:Consurf_key_small.gif|200px|right]]<br />
Check<jmol><br />
<jmolCheckbox><br />
<scriptWhenChecked>; select protein; define ~consurf_to_do selected; consurf_initial_scene = true; script "/wiki/ConSurf/d6/1d66_consurf.spt"</scriptWhenChecked><br />
<scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/initialview01.spt</scriptWhenUnchecked><br />
<text>to colour the structure by Evolutionary Conservation</text><br />
</jmolCheckbox><br />
</jmol>, as determined by [http://consurfdb.tau.ac.il/ ConSurfDB]. You may read the [[Conservation%2C_Evolutionary|explanation]] of the method and the full data available from [http://bental.tau.ac.il/new_ConSurfDB/main_output.php?pdb_ID=1d66 ConSurf].<br />
<div style="clear:both"></div><br />
<div style="background-color:#fffaf0;"><br />
== Publication Abstract from PubMed ==<br />
A specific DNA complex of the 65-residue, N-terminal fragment of the yeast transcriptional activator, GAL4, has been analysed at 2.7 A resolution by X-ray crystallography. The protein binds as a <scene name='92/925538/Dimer/2'>dimer</scene> to a symmetrical 17-base-pair sequence. There is a compact <scene name='92/925538/Metal_binding_domain/5'>metal binding domain</scene> (residues 8-40), an <scene name='92/925538/Extended_linker/4'>extended linker</scene> (41-49), and an <scene name='92/925538/Alpha-helical_dimerization/2'>alpha-helical dimerization element</scene> (50-64). A small, Zn(2+)-containing domain recognizes a conserved CCG triplet at each end of the site through direct contacts with the major groove. The metal binding domain contains <scene name='92/925538/Cysteine_metal_binding/3'>cysteine residues</scene> that coordinate to the metal as shown as cadmium. A short coiled-coil dimerization element imposes 2-fold symmetry. A segment of extended polypeptide chain links the metal-binding module to the dimerization element and specifies the length of the site. The relatively open structure of the complex would allow another protein to bind coordinately with GAL4.<br />
<br />
DNA recognition by GAL4: structure of a protein-DNA complex.,Marmorstein R, Carey M, Ptashne M, Harrison SC Nature. 1992 Apr 2;356(6368):408-14. PMID:1557122<ref>PMID:1557122</ref><br />
<br />
From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br><br />
</div><br />
<div class="pdbe-citations 1d66" style="background-color:#fffaf0;"></div><br />
<br />
==See Also==<br />
*[[Gal3-Gal80-Gal4|Gal3-Gal80-Gal4]]<br />
*[[Hydrogen in macromolecular models|Hydrogen in macromolecular models]]<br />
== References ==<br />
<references/><br />
__TOC__<br />
</StructureSection><br />
[[Category: Atcc 18824]]<br />
[[Category: Large Structures]]<br />
[[Category: Carey, M]]<br />
[[Category: Harrison, S C]]<br />
[[Category: Marmorstein, R]]<br />
[[Category: Ptashne, M]]<br />
[[Category: Double helix]]<br />
[[Category: Protein-dna complex]]<br />
[[Category: Transcription-dna complex]]<br />
<br />
==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH gRNA==<br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA rendering it inert. <scene name='92/925538/4un3/3'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single stranded guide RNA (sgRNA) to bind the target DNA that will be cut. Cas9 utalizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind to Cas9 to be cut. The PAM sequence acts as a two factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. The main domains in the Cas9 are the REC lobe (residues 41–425) and NUC lobe (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WEB, and PI domains (1). The REC lobe is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site (2). The RuvC nuclease domain(residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the sgRNA (3). The HNH domain (residues 520–628) cleaves the target strand of DNA bound to the sgRNA (3). The WEB domain (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of twisted five-stranded beta sheet flanked by four alpha helices (4). The PI domain (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA (5). There are also two linker domains, L1 (residues 481–519) and L2 (residues 629–649), that connect the RuvC and HNH (1). Furthermore, there is a phosphate lock loop (residues 775–787) that connect the WEB and RuvC domains. Cas9 has four main mechanisms that are important for successful cleavage including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is a the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe interacts with the seed region of the sgRNA (C13-C20) as well as the PAM distal region (A3-U6) through the phosphate backbone. The seed region is in the A-form confirmation so it can bind the target DNA. The target DNA binds to the REC loop and RuvC domain for the proper conformation for base paring between the target DNA and sgRNA. <br />
== Recognition of the PAM seequence ==<br />
For the recognition of the PAM sequence, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex. <br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the REC and WED domains. The WED domain contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat:anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, RuvC and HNH are in endonuclease activity. RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA.<br />
<br />
== Citations ==<br />
1. Hiroshi Nishimasu, Le Cong, Winston X. Yan, F. Ann Ran, Bernd Zetsche, Yinqing Li, Arisa Kurabayashi, Ryuichiro Ishitani, Feng Zhang, Osamu Nureki, Crystal Structure of Staphylococcus aureus Cas9, Cell,Volume 162, Issue 5,2015,Pages 1113-1126,ISSN 0092-8674,https://doi.org/10.1016/j.cell.2015.08.007.<br />
<br />
2. Palermo G, Chen JS, Ricci CG, Rivalta I, Jinek M, Batista VS, Doudna JA, McCammon JA. Key role of the REC lobe during CRISPR-Cas9 activation by 'sensing', 'regulating', and 'locking' the catalytic HNH domain. Q Rev Biophys. 2018;51:e91. doi: 10.1017/S0033583518000070. Epub 2018 Aug 3. PMID: 30555184; PMCID: PMC6292676.<br />
<br />
3. Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani R, Zhang F, Nureki O. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell. 2014 Feb 27;156(5):935-49. doi: 10.1016/j.cell.2014.02.001. Epub 2014 Feb 13. PMID: 24529477; PMCID: PMC4139937.<br />
<br />
4. Morlot C, Pernot L, Le Gouellec A, Di Guilmi AM, Vernet T, Dideberg O, Dessen A. Crystal structure of a peptidoglycan synthesis regulatory factor (PBP3) from Streptococcus pneumoniae. J Biol Chem. 2005 Apr 22;280(16):15984-91. doi: 10.1074/jbc.M408446200. Epub 2004 Dec 13. PMID: 15596446.<br />
<br />
5. Chen H, Choi J, Bailey S. Cut site selection by the two nuclease domains of the Cas9 RNA-guided endonuclease. J Biol Chem. 2014 May 9;289(19):13284-94. doi: 10.1074/jbc.M113.539726. Epub 2014 Mar 14. PMID: 24634220; PMCID: PMC4036338.</div>Thomas Gastineauhttps://proteopedia.org/index.php?title=Sandbox_Reserved_1750&diff=3641348Sandbox Reserved 17502022-10-04T14:59:49Z<p>Thomas Gastineau: </p>
<hr />
<div>Displayed</scene></scene>==DNA RECOGNITION BY GAL4: STRUCTURE OF A PROTEIN/DNA COMPLEX==<br />
<StructureSection load='1d66' size='340' side='right'caption='[[1d66]], [[Resolution|resolution]] 2.70&Aring;' scene=''><br />
== Structural highlights ==<br />
<table><tr><td colspan='2'>[[1d66]] is a 4 chain structure with sequence from [https://en.wikipedia.org/wiki/Atcc_18824 Atcc 18824]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1D66 OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=1D66 FirstGlance]. <br><br />
</td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=CD:CADMIUM+ION'>CD</scene></td></tr><br />
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=1d66 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1d66 OCA], [https://pdbe.org/1d66 PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=1d66 RCSB], [https://www.ebi.ac.uk/pdbsum/1d66 PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=1d66 ProSAT]</span></td></tr><br />
</table><br />
== Function ==<br />
[[https://www.uniprot.org/uniprot/GAL4_YEAST GAL4_YEAST]] This protein is a positive regulator for the gene expression of the galactose-induced genes such as GAL1, GAL2, GAL7, GAL10, and MEL1 which code for the enzymes used to convert galactose to glucose. It recognizes a 17 base pair sequence in (5'-CGGRNNRCYNYNCNCCG-3') the upstream activating sequence (UAS-G) of these genes. This sequence is correctly shown on the structure as (5'-CGGAGGACTGCCCTCCG-3'). <scene name='92/925538/Dna_protein_interaction/3'>The DNA-Protein Interaction</scene> with all of the base pairs within 5 angstroms of the protein highlighted illustrates that the protein interacts with both strands of the UAS.<br />
== Evolutionary Conservation ==<br />
[[Image:Consurf_key_small.gif|200px|right]]<br />
Check<jmol><br />
<jmolCheckbox><br />
<scriptWhenChecked>; select protein; define ~consurf_to_do selected; consurf_initial_scene = true; script "/wiki/ConSurf/d6/1d66_consurf.spt"</scriptWhenChecked><br />
<scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/initialview01.spt</scriptWhenUnchecked><br />
<text>to colour the structure by Evolutionary Conservation</text><br />
</jmolCheckbox><br />
</jmol>, as determined by [http://consurfdb.tau.ac.il/ ConSurfDB]. You may read the [[Conservation%2C_Evolutionary|explanation]] of the method and the full data available from [http://bental.tau.ac.il/new_ConSurfDB/main_output.php?pdb_ID=1d66 ConSurf].<br />
<div style="clear:both"></div><br />
<div style="background-color:#fffaf0;"><br />
== Publication Abstract from PubMed ==<br />
A specific DNA complex of the 65-residue, N-terminal fragment of the yeast transcriptional activator, GAL4, has been analysed at 2.7 A resolution by X-ray crystallography. The protein binds as a <scene name='92/925538/Dimer/2'>dimer</scene> to a symmetrical 17-base-pair sequence. There is a compact <scene name='92/925538/Metal_binding_domain/5'>metal binding domain</scene> (residues 8-40), an <scene name='92/925538/Extended_linker/4'>extended linker</scene> (41-49), and an <scene name='92/925538/Alpha-helical_dimerization/2'>alpha-helical dimerization element</scene> (50-64). A small, Zn(2+)-containing domain recognizes a conserved CCG triplet at each end of the site through direct contacts with the major groove. The metal binding domain contains <scene name='92/925538/Cysteine_metal_binding/3'>cysteine residues</scene> that coordinate to the metal as shown as cadmium. A short coiled-coil dimerization element imposes 2-fold symmetry. A segment of extended polypeptide chain links the metal-binding module to the dimerization element and specifies the length of the site. The relatively open structure of the complex would allow another protein to bind coordinately with GAL4.<br />
<br />
DNA recognition by GAL4: structure of a protein-DNA complex.,Marmorstein R, Carey M, Ptashne M, Harrison SC Nature. 1992 Apr 2;356(6368):408-14. PMID:1557122<ref>PMID:1557122</ref><br />
<br />
From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br><br />
</div><br />
<div class="pdbe-citations 1d66" style="background-color:#fffaf0;"></div><br />
<br />
==See Also==<br />
*[[Gal3-Gal80-Gal4|Gal3-Gal80-Gal4]]<br />
*[[Hydrogen in macromolecular models|Hydrogen in macromolecular models]]<br />
== References ==<br />
<references/><br />
__TOC__<br />
</StructureSection><br />
[[Category: Atcc 18824]]<br />
[[Category: Large Structures]]<br />
[[Category: Carey, M]]<br />
[[Category: Harrison, S C]]<br />
[[Category: Marmorstein, R]]<br />
[[Category: Ptashne, M]]<br />
[[Category: Double helix]]<br />
[[Category: Protein-dna complex]]<br />
[[Category: Transcription-dna complex]]<br />
<br />
==STRUCTURE OF Cas9 IN STAPHYLOCOCCUS AUREUS IN COMPLEX WITH gRNA==<br />
== Cas9 Overview ==<br />
CRISPR is a bacterial immune response to bacteriophages to prevent subsequent infections and is a form of acquired immunity. Within the CRISPR system, Cas9 is a protein responsible for cutting the viral DNA rendering it inert. <scene name='92/925538/4un3/3'>Cas9</scene> structure in Staphylococcus aureus (SaCas9) utilizes a single stranded guide RNA (sgRNA) to bind the target DNA that will be cut. Cas9 utalizes the sgRNA as an RNA guide to cut the target DNA sequence and binds complimentary to target DNA so Cas9 create a double stranded DNA break in the proper location. The target DNA must also have a PAM sequence to bind to Cas9 to be cut. The PAM sequence acts as a two factor authentication in junction with the sgRNA that tells the Cas9 to cut this portion of DNA. The main domains in the Cas9 are the REC lobe (residues 41–425) and NUC lobe (residues 1–40 and 435–1053). These lobes are connected by an arginine rich bridge helix (residues 41–73) and a linker loop (residues 426–434). The NUC lobe contains RuvC, HNH, WEB, and PI domains (1). The REC lobe is responsible for recognizing the nucleic acids present causing a conformational change in the HNH locking the HNH into the cleavage site (2). The RuvC nuclease domain(residues 1–40, 435–480 and 650–774) cleaves the strand of target DNA that is not bound complimentary to the sgRNA (3). The HNH domain cleaves the target strand of DNA bound to the sgRNA (3). The WEB domain (residues 788–909) is responsible for recognizing the sgRNA scaffold and consists of twisted five-stranded beta sheet flanked by four alpha helices (4). The PI domain (residues 910–1053) recognizes the PAM sequence on the target DNA that is not complimentary to the sgRNA (5). There are also two linker domains, L1 (residues 481–519) and L2 (residues 629–649) that connect the RuvC and HNH (1). Furthermore, there is a phosphate lock loop (residues 775–787) that connect the WEB and RuvC domains. Cas9 has four main mechanisms that are important for successful cleavage including recognition of the sgRNA-target heteroduplex, recognition of the PAM sequence, recognition of the sgRNA scaffold, and endonuclease activity by HNH and RuvC.<br />
== Recognition of the sgRNA-target heteroduplex ==<br />
The recognition of the sgRNA-target heteroduplex in Cas9 begins by inserting itself into the central channel between the REC and NUC lobes. A heteroduplex is a the binding of the complimentary strands of the sgRNA and target DNA. The REC lobe interacts with the seed region of the sgRNA (C13-C20) as well as the PAM distal region (A3-U6) through the phosphate backbone. The seed region is in the A-form confirmation so it can bind the target DNA. The target DNA binds to the REC loop and RuvC domain for the proper conformation for base paring between the target DNA and sgRNA. <br />
== Recognition of the PAM seequence ==<br />
For the recognition of the PAM sequence, the target DNA with the PAM sequence (5’-NNGRRN-3’) is bound to SaCas9 through bidentate hydrogen bonds as well as direct and water mediated hydrogen bonds through the major groove in the PI domain. The WED domain recognizes the minor groove phosphate backbone of the duplex. <br />
== Recognition of the sgRNA scaffold ==<br />
The SaCas9 recognizes the sgRNA scaffold within the REC and WED domains. The WED domain contains five stranded beta sheets flanked with four alpha helices to allow binding of the repeat:anti-repeat duplex. REC lob binds the scaffold and secures it into the SaCas9.<br />
== Endonuclease Activity of Cas9 ==<br />
Finally, RuvC and HNH are in endonuclease activity. RuvC uses a two-metal ion mechanism of manganese to cleave the non-target DNA and causes a conformational change in L1. This conformational change leads to the phosphate group of the target strand to be cleaved by HNH. HNH includes a beta beta alpha metal fold and uses a one metal ion mechanism to cleave the target DNA.<br />
<br />
== Citations ==<br />
1. Hiroshi Nishimasu, Le Cong, Winston X. Yan, F. Ann Ran, Bernd Zetsche, Yinqing Li, Arisa Kurabayashi, Ryuichiro Ishitani, Feng Zhang, Osamu Nureki, Crystal Structure of Staphylococcus aureus Cas9, Cell,Volume 162, Issue 5,2015,Pages 1113-1126,ISSN 0092-8674,https://doi.org/10.1016/j.cell.2015.08.007.<br />
<br />
2. Palermo G, Chen JS, Ricci CG, Rivalta I, Jinek M, Batista VS, Doudna JA, McCammon JA. Key role of the REC lobe during CRISPR-Cas9 activation by 'sensing', 'regulating', and 'locking' the catalytic HNH domain. Q Rev Biophys. 2018;51:e91. doi: 10.1017/S0033583518000070. Epub 2018 Aug 3. PMID: 30555184; PMCID: PMC6292676.<br />
<br />
3. Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani R, Zhang F, Nureki O. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell. 2014 Feb 27;156(5):935-49. doi: 10.1016/j.cell.2014.02.001. Epub 2014 Feb 13. PMID: 24529477; PMCID: PMC4139937.<br />
<br />
4. Morlot C, Pernot L, Le Gouellec A, Di Guilmi AM, Vernet T, Dideberg O, Dessen A. Crystal structure of a peptidoglycan synthesis regulatory factor (PBP3) from Streptococcus pneumoniae. J Biol Chem. 2005 Apr 22;280(16):15984-91. doi: 10.1074/jbc.M408446200. Epub 2004 Dec 13. PMID: 15596446.<br />
<br />
5. Chen H, Choi J, Bailey S. Cut site selection by the two nuclease domains of the Cas9 RNA-guided endonuclease. J Biol Chem. 2014 May 9;289(19):13284-94. doi: 10.1074/jbc.M113.539726. Epub 2014 Mar 14. PMID: 24634220; PMCID: PMC4036338.</div>Thomas Gastineau