View source for 9mku


==FnoCas12a bridge helix variant state 2==
<StructureSection load='9mku' size='340' side='right'caption='[[9mku]], [[Resolution|resolution]] 4.00&Aring;' scene=''>
== Structural highlights ==
<table><tr><td colspan='2'>[[9mku]] is a 3 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=9MKU OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=9MKU FirstGlance]. <br>
</td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">Electron Microscopy, [[Resolution|Resolution]] 4&#8491;</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=9mku FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=9mku OCA], [https://pdbe.org/9mku PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=9mku RCSB], [https://www.ebi.ac.uk/pdbsum/9mku PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=9mku 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 ==
CRISPR-Cas12a, an RNA-based DNA targeting system, is widely used for genome editing and biomarker detection. To mitigate the off-target DNA cleavage of Cas12a, we previously developed a Francisella novicida Cas12a variant (FnoCas12a(KD2P)) by introducing double proline substitutions (K969P/D970P) in a conserved arginine-rich helix called the bridge helix (BH). In this work, we use a combinatorial approach to understand the molecular mechanisms of BH-mediated activation of Cas12a for DNA cleavage. We report five structures of FnoCas12a(KD2P) that are at different states of conformational activation. Comparison of the variant and wild-type (FnoCas12a(WT)) structures, along with activity assays and computational simulations, establishes the loop-to-helical transition and bending of the BH as an allosteric trigger for RNA-DNA hybrid propagation. These changes track with the previously reported coupled remodeling of BH and helix 1 of RuvC motif-II as well as the REC lobe movements needed to accommodate the growing hybrid. The transition of the BH is essential for the loop-to-helical transition of the "lid", which in turn opens the RuvC active site pocket for DNA entry and cleavage. Pairwise 3D structural comparison of the BH and RuvC of Cas12 and Cas9 families provides insight into the diversity of BH's structural organization in these mechanistically similar enzymes.

Bridge helix of Cas12a is an allosteric regulator of R-loop formation and RuvC activation.,Ganguly C, Aribam SD, Dos Santos AM, Martin L, Thomas LM, Shao Y, Rajan R Nat Commun. 2026 Jan 28. doi: 10.1038/s41467-026-68657-0. PMID:41605928<ref>PMID:41605928</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 9mku" style="background-color:#fffaf0;"></div>
== References ==
<references/>
__TOC__
</StructureSection>
[[Category: Francisella tularensis subsp. novicida U112]]
[[Category: Large Structures]]
[[Category: Synthetic construct]]
[[Category: Aribam SD]]
[[Category: Ganguly C]]
[[Category: Martin L]]
[[Category: Rajan R]]
[[Category: Thomas LM]]