7s3h: Difference between revisions
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The | ==Cas9:sgRNA:DNA (S. pyogenes) with 0 RNA:DNA base pairs, open-protein/linear-DNA conformation== | ||
<StructureSection load='7s3h' size='340' side='right'caption='[[7s3h]], [[Resolution|resolution]] 2.50Å' scene=''> | |||
== Structural highlights == | |||
<table><tr><td colspan='2'>[[7s3h]] is a 4 chain structure with sequence from [https://en.wikipedia.org/wiki/Streptococcus_pyogenes Streptococcus pyogenes], [https://en.wikipedia.org/wiki/Streptococcus_pyogenes_serotype_M1 Streptococcus pyogenes serotype M1] 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=7S3H OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=7S3H 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]] 2.5Å</td></tr> | |||
<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=2YR:2-DEOXY-N-(2-SULFANYLETHYL)CYTIDINE+5-(DIHYDROGEN+PHOSPHATE)'>2YR</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=7s3h FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=7s3h OCA], [https://pdbe.org/7s3h PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=7s3h RCSB], [https://www.ebi.ac.uk/pdbsum/7s3h PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=7s3h ProSAT]</span></td></tr> | |||
</table> | |||
== Function == | |||
[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 == | |||
In bacterial defense and genome editing applications, the CRISPR-associated protein Cas9 searches millions of DNA base pairs to locate a 20-nucleotide, guide RNA-complementary target sequence that abuts a protospacer-adjacent motif (PAM). Target capture requires Cas9 to unwind DNA at candidate sequences using an unknown ATP-independent mechanism. Here we show that Cas9 sharply bends and undertwists DNA on PAM binding, thereby flipping DNA nucleotides out of the duplex and toward the guide RNA for sequence interrogation. Cryogenic-electron microscopy (cryo-EM) structures of Cas9-RNA-DNA complexes trapped at different states of the interrogation pathway, together with solution conformational probing, reveal that global protein rearrangement accompanies formation of an unstacked DNA hinge. Bend-induced base flipping explains how Cas9 'reads' snippets of DNA to locate target sites within a vast excess of nontarget DNA, a process crucial to both bacterial antiviral immunity and genome editing. This mechanism establishes a physical solution to the problem of complementarity-guided DNA search and shows how interrogation speed and local DNA geometry may influence genome editing efficiency. | |||
CRISPR-Cas9 bends and twists DNA to read its sequence.,Cofsky JC, Soczek KM, Knott GJ, Nogales E, Doudna JA Nat Struct Mol Biol. 2022 Apr;29(4):395-402. doi: 10.1038/s41594-022-00756-0. , Epub 2022 Apr 14. PMID:35422516<ref>PMID:35422516</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
[[Category: | </div> | ||
[[Category: | <div class="pdbe-citations 7s3h" style="background-color:#fffaf0;"></div> | ||
[[Category: Cofsky | |||
[[Category: Knott | ==See Also== | ||
[[Category: | *[[Endonuclease 3D structures|Endonuclease 3D structures]] | ||
[[Category: Soczek | == References == | ||
<references/> | |||
__TOC__ | |||
</StructureSection> | |||
[[Category: Large Structures]] | |||
[[Category: Streptococcus pyogenes]] | |||
[[Category: Streptococcus pyogenes serotype M1]] | |||
[[Category: Synthetic construct]] | |||
[[Category: Cofsky JC]] | |||
[[Category: Doudna JA]] | |||
[[Category: Knott GJ]] | |||
[[Category: Nogales E]] | |||
[[Category: Soczek KM]] | |||
Latest revision as of 16:59, 6 November 2024
Cas9:sgRNA:DNA (S. pyogenes) with 0 RNA:DNA base pairs, open-protein/linear-DNA conformationCas9:sgRNA:DNA (S. pyogenes) with 0 RNA:DNA base pairs, open-protein/linear-DNA conformation
Structural highlights
FunctionCAS9_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.[1] [2] Publication Abstract from PubMedIn bacterial defense and genome editing applications, the CRISPR-associated protein Cas9 searches millions of DNA base pairs to locate a 20-nucleotide, guide RNA-complementary target sequence that abuts a protospacer-adjacent motif (PAM). Target capture requires Cas9 to unwind DNA at candidate sequences using an unknown ATP-independent mechanism. Here we show that Cas9 sharply bends and undertwists DNA on PAM binding, thereby flipping DNA nucleotides out of the duplex and toward the guide RNA for sequence interrogation. Cryogenic-electron microscopy (cryo-EM) structures of Cas9-RNA-DNA complexes trapped at different states of the interrogation pathway, together with solution conformational probing, reveal that global protein rearrangement accompanies formation of an unstacked DNA hinge. Bend-induced base flipping explains how Cas9 'reads' snippets of DNA to locate target sites within a vast excess of nontarget DNA, a process crucial to both bacterial antiviral immunity and genome editing. This mechanism establishes a physical solution to the problem of complementarity-guided DNA search and shows how interrogation speed and local DNA geometry may influence genome editing efficiency. CRISPR-Cas9 bends and twists DNA to read its sequence.,Cofsky JC, Soczek KM, Knott GJ, Nogales E, Doudna JA Nat Struct Mol Biol. 2022 Apr;29(4):395-402. doi: 10.1038/s41594-022-00756-0. , Epub 2022 Apr 14. PMID:35422516[3] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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