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| | ==Structure of nuclease in complex with associated protein== | | ==Structure of nuclease in complex with associated protein== |
| - | <StructureSection load='5xbl' size='340' side='right' caption='[[5xbl]], [[Resolution|resolution]] 3.05Å' scene=''> | + | <StructureSection load='5xbl' size='340' side='right'caption='[[5xbl]], [[Resolution|resolution]] 3.05Å' scene=''> |
| | == Structural highlights == | | == Structural highlights == |
| - | <table><tr><td colspan='2'>[[5xbl]] is a 3 chain structure with sequence from [http://en.wikipedia.org/wiki/"bacterium_monocytogenes_hominis"_nyfeldt_1932 "bacterium monocytogenes hominis" nyfeldt 1932] and [http://en.wikipedia.org/wiki/Strp1 Strp1]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=5XBL OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=5XBL FirstGlance]. <br> | + | <table><tr><td colspan='2'>[[5xbl]] is a 3 chain structure with sequence from [https://en.wikipedia.org/wiki/Listeria_monocytogenes Listeria monocytogenes], [https://en.wikipedia.org/wiki/Streptococcus_pyogenes Streptococcus pyogenes] and [https://en.wikipedia.org/wiki/Streptococcus_pyogenes_serotype_M1 Streptococcus pyogenes serotype M1]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=5XBL OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=5XBL FirstGlance]. <br> |
| - | </td></tr><tr id='gene'><td class="sblockLbl"><b>[[Gene|Gene:]]</b></td><td class="sblockDat">cas9, csn1, SPy_1046 ([http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=301447 STRP1])</td></tr> | + | </td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 3.052Å</td></tr> |
| - | <tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=5xbl FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=5xbl OCA], [http://pdbe.org/5xbl PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=5xbl RCSB], [http://www.ebi.ac.uk/pdbsum/5xbl PDBsum], [http://prosat.h-its.org/prosat/prosatexe?pdbcode=5xbl ProSAT]</span></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=5xbl FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=5xbl OCA], [https://pdbe.org/5xbl PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=5xbl RCSB], [https://www.ebi.ac.uk/pdbsum/5xbl PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=5xbl ProSAT]</span></td></tr> |
| | </table> | | </table> |
| | == Function == | | == Function == |
| - | [[http://www.uniprot.org/uniprot/CAS9_STRP1 CAS9_STRP1]] 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 spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA) (Probable). In type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and this protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed by 3'-5' exonucleolytically. DNA-binding requires protein and both RNA species. Cas9 probably recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus nonself.<ref>PMID:21455174</ref> <ref>PMID:22745249</ref> | + | [https://www.uniprot.org/uniprot/CAS9_STRP1 CAS9_STRP1] 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 spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA) (Probable). In type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and this protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed by 3'-5' exonucleolytically. DNA-binding requires protein and both RNA species. Cas9 probably recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus nonself.<ref>PMID:21455174</ref> <ref>PMID:22745249</ref> |
| - | <div style="background-color:#fffaf0;">
| + | |
| - | == Publication Abstract from PubMed ==
| + | |
| - | CRISPR-Cas9 systems are bacterial adaptive immune systems that defend against infection by phages. Through the RNA-guided endonuclease activity of Cas9 they degrade double-stranded DNA with a protospacer adjacent motif (PAM) and sequences complementary to the guide RNA. Recently, two anti-CRISPR proteins (AcrIIA2 and AcrIIA4 from Listeria monocytogenes prophages) were identified, both of which inhibit Streptococcus pyogenes Cas9 (SpyCas9) and L. monocytogenes Cas9 activity in bacteria and human cells. However, the mechanism of AcrIIA2- or AcrIIA4-mediated Cas9 inhibition remains unknown. Here we report a crystal structure of SpyCas9 in complex with a single-guide RNA (sgRNA) and AcrIIA4. Our data show that AcrIIA2 and AcrIIA4 interact with SpyCas9 in a sgRNA-dependent manner. The structure reveals that AcrIIA4 inhibits SpyCas9 activity by structurally mimicking the PAM to occupy the PAM-interacting site in the PAM-interacting domain, thereby blocking recognition of double-stranded DNA substrates by SpyCas9. AcrIIA4 further inhibits the endonuclease activity of SpyCas9 by shielding its RuvC active site. Structural comparison reveals that formation of the AcrIIA4-binding site of SpyCas9 is induced by sgRNA binding. Our study reveals the mechanism of SpyCas9 inhibition by AcrIIA4, providing a structural basis for developing 'off-switch' tools for SpyCas9 to avoid unwanted genome edits within cells and tissues.
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| - | Structural basis of CRISPR-SpyCas9 inhibition by an anti-CRISPR protein.,Dong, Guo M, Wang S, Zhu Y, Wang S, Xiong Z, Yang J, Xu Z, Huang Z Nature. 2017 Jun 15;546(7658):436-439. doi: 10.1038/nature22377. Epub 2017 Apr, 27. PMID:28448066<ref>PMID:28448066</ref>
| + | ==See Also== |
| - | | + | *[[Endonuclease 3D structures|Endonuclease 3D structures]] |
| - | From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br>
| + | |
| - | </div>
| + | |
| - | <div class="pdbe-citations 5xbl" style="background-color:#fffaf0;"></div>
| + | |
| | == References == | | == References == |
| | <references/> | | <references/> |
| | __TOC__ | | __TOC__ |
| | </StructureSection> | | </StructureSection> |
| - | [[Category: Bacterium monocytogenes hominis nyfeldt 1932]] | + | [[Category: Large Structures]] |
| - | [[Category: Strp1]] | + | [[Category: Listeria monocytogenes]] |
| - | [[Category: Dong, D]] | + | [[Category: Streptococcus pyogenes]] |
| - | [[Category: Guo, M]] | + | [[Category: Streptococcus pyogenes serotype M1]] |
| - | [[Category: Huang, Z]] | + | [[Category: Dong D]] |
| - | [[Category: Wang, S]] | + | [[Category: Guo M]] |
| - | [[Category: Zhu, Y]] | + | [[Category: Huang Z]] |
| - | [[Category: Hydrolase-rna complex]] | + | [[Category: Wang S]] |
| - | [[Category: Nuclease]] | + | [[Category: Zhu Y]] |
