View source for 5nfv


==Crystal structure of catalytically inactive FnCas12 mutant bound to an R-loop structure containing a pre-crRNA mimic and full-length DNA target==
<StructureSection load='5nfv' size='340' side='right'caption='[[5nfv]], [[Resolution|resolution]] 2.50&Aring;' scene=''>
== Structural highlights ==
<table><tr><td colspan='2'>[[5nfv]] is a 4 chain structure with sequence from [https://en.wikipedia.org/wiki/Francisella_tularensis_subsp._novicida_U112 Francisella tularensis subsp. novicida U112] and [https://en.wikipedia.org/wiki/Synthetic_construct Synthetic construct]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=5NFV OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=5NFV FirstGlance]. <br>
</td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 2.501&#8491;</td></tr>
<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=B3P:2-[3-(2-HYDROXY-1,1-DIHYDROXYMETHYL-ETHYLAMINO)-PROPYLAMINO]-2-HYDROXYMETHYL-PROPANE-1,3-DIOL'>B3P</scene>, <scene name='pdbligand=MG:MAGNESIUM+ION'>MG</scene></td></tr>
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=5nfv FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=5nfv OCA], [https://pdbe.org/5nfv PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=5nfv RCSB], [https://www.ebi.ac.uk/pdbsum/5nfv PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=5nfv ProSAT]</span></td></tr>
</table>
== Function ==
[https://www.uniprot.org/uniprot/CS12A_FRATN CS12A_FRATN] CRISPR (clustered regularly interspaced short palindromic repeat), is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain sequences complementary to antecedent mobile elements and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). Has endonuclease activity on pre-crRNA and dsDNA, using different active sites. A single-RNA guided endonuclease that is also capable of guiding crRNA processing; correct processing of pre-crRNA requires only this protein and the CRISPR locus (PubMed:26422227, PubMed:27096362). pre-crRNA processing proceeds by an intramolecular nucleophilic attack on the scissile phosphate by the 2'-OH of the upstream ribonucleotide, the divalent cation (which is bound by the crRNA) is probably required for ordering the crRNA pseudoknot and/or increasing RNA binding (PubMed:28431230). RNA mutagenesis studies show pre-crRNA cleavage is highly sequence- and structure-specific (PubMed:27096362). Forms a complex with crRNA and complementary dsDNA, where the crRNA displaces the non-target DNA strand and directs endonucleolytic cleavage of both strands of the DNA (PubMed:26422227, PubMed:27096362, PubMed:28431230). Cleavage results in staggered 5-base 5' overhangs 14-18 and 21-23 bases downstream of the PAM (protospacer adjacent motif) on the non-target and target strands respectively (PubMed:26422227, PubMed:28431230, PubMed:28562584). Both target and non-target strand DNA are probably independently cleaved in the same active site (PubMed:28431230, PubMed:28562584). When this protein is expressed in E.coli it prevents plasmids homologous to the first CRISPR spacer from transforming, formally showing it is responsible for plasmid immunity (PubMed:26422227).<ref>PMID:26422227</ref> <ref>PMID:27096362</ref> <ref>PMID:28431230</ref> <ref>PMID:28562584</ref> 
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== Publication Abstract from PubMed ==
The CRISPR-associated protein Cas12a (Cpf1), which has been repurposed for genome editing, possesses two distinct nuclease activities: endoribonuclease activity for processing its own guide RNAs and RNA-guided DNase activity for target DNA cleavage. To elucidate the molecular basis of both activities, we determined crystal structures of Francisella novicida Cas12a bound to guide RNA and in complex with an R-loop formed by a non-cleavable guide RNA precursor and a full-length target DNA. Corroborated by biochemical experiments, these structures reveal the mechanisms of guide RNA processing and pre-ordering of the seed sequence in the guide RNA that primes Cas12a for target DNA binding. Furthermore, the R-loop complex structure reveals the strand displacement mechanism that facilitates guide-target hybridization and suggests a mechanism for double-stranded DNA cleavage involving a single active site. Together, these insights advance our mechanistic understanding of Cas12a enzymes and may contribute to further development of genome editing technologies.

Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a.,Swarts DC, van der Oost J, Jinek M Mol Cell. 2017 Apr 20;66(2):221-233.e4. doi: 10.1016/j.molcel.2017.03.016. PMID:28431230<ref>PMID:28431230</ref>

From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br>
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<div class="pdbe-citations 5nfv" style="background-color:#fffaf0;"></div>

==See Also==
*[[Endonuclease 3D structures|Endonuclease 3D structures]]
== References ==
<references/>
__TOC__
</StructureSection>
[[Category: Francisella tularensis subsp. novicida U112]]
[[Category: Large Structures]]
[[Category: Synthetic construct]]
[[Category: Jinek M]]
[[Category: Swarts DC]]
[[Category: Van der Oost J]]