7vw3: Difference between revisions
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The | ==Cryo-EM structure of SaCas9-sgRNA-DNA ternary complex== | ||
<StructureSection load='7vw3' size='340' side='right'caption='[[7vw3]], [[Resolution|resolution]] 3.80Å' scene=''> | |||
== Structural highlights == | |||
<table><tr><td colspan='2'>[[7vw3]] is a 4 chain structure with sequence from [https://en.wikipedia.org/wiki/Staphylococcus_aureus Staphylococcus aureus] 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=7VW3 OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=7VW3 FirstGlance]. <br> | |||
</td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><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=7vw3 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=7vw3 OCA], [https://pdbe.org/7vw3 PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=7vw3 RCSB], [https://www.ebi.ac.uk/pdbsum/7vw3 PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=7vw3 ProSAT]</span></td></tr> | |||
</table> | |||
== Function == | |||
[https://www.uniprot.org/uniprot/CAS9_STAAU CAS9_STAAU] 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). 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; Cas9 is inactive in the absence of the 2 guide RNAs (gRNA). Cas9 recognizes the protospacer adjacent motif (PAM) in the CRISPR repeat sequences to help distinguish self versus nonself, as targets within the bacterial CRISPR locus do not have PAMs. PAM recognition is also required for catalytic activity.[HAMAP-Rule:MF_01480]<ref>PMID:25830891</ref> <ref>PMID:26098369</ref> | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
Staphylococcus aureus Cas9 (SaCas9) is a widely used genome editing tool. Understanding its molecular mechanisms of DNA cleavage could effectively guide the engineering optimization of this system. Here, we determined the first cryo-electron microscopy structure of the SaCas9-sgRNA-DNA ternary complex. This structure reveals that the HNH nuclease domain is tightly bound to the cleavage site of the target DNA strand, and is in close contact with the WED and REC domains. Moreover, it captures the complete structure of the sgRNA, including the previously unresolved stem-loop 2. Based on this structure, we build a full-length model for the ternary complex in cleavage state. This model enables identification of the residues for the interactions between the HNH domain and the WED and REC domains. Moreover, we found that the stem-loop 2 of the sgRNA tightly binds to the PI and RuvC domains and may also regulate the position shift of the RuvC domain. Further mutagenesis and molecular dynamics simulations supported the idea that the interactions of the HNH domain with the WED and REC domains play an important role in the DNA cleavage. Thus, this study provides new mechanistic insights into the DNA cleavage of SaCas9 and is also useful for guiding the future engineering of SaCas9-mediated gene editing systems. | |||
Full-Length Model of SaCas9-sgRNA-DNA Complex in Cleavage State.,Du W, Zhu H, Qian J, Xue D, Zheng S, Huang Q Int J Mol Sci. 2023 Jan 7;24(2):1204. doi: 10.3390/ijms24021204. PMID:36674715<ref>PMID:36674715</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
[[Category: | </div> | ||
[[Category: Du | <div class="pdbe-citations 7vw3" style="background-color:#fffaf0;"></div> | ||
[[Category: Huang | == References == | ||
[[Category: Qian | <references/> | ||
__TOC__ | |||
</StructureSection> | |||
[[Category: Large Structures]] | |||
[[Category: Staphylococcus aureus]] | |||
[[Category: Synthetic construct]] | |||
[[Category: Du WH]] | |||
[[Category: Huang Q]] | |||
[[Category: Qian JQ]] | |||
Latest revision as of 12:48, 15 March 2023
Cryo-EM structure of SaCas9-sgRNA-DNA ternary complexCryo-EM structure of SaCas9-sgRNA-DNA ternary complex
Structural highlights
FunctionCAS9_STAAU 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). 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; Cas9 is inactive in the absence of the 2 guide RNAs (gRNA). Cas9 recognizes the protospacer adjacent motif (PAM) in the CRISPR repeat sequences to help distinguish self versus nonself, as targets within the bacterial CRISPR locus do not have PAMs. PAM recognition is also required for catalytic activity.[HAMAP-Rule:MF_01480][1] [2] Publication Abstract from PubMedStaphylococcus aureus Cas9 (SaCas9) is a widely used genome editing tool. Understanding its molecular mechanisms of DNA cleavage could effectively guide the engineering optimization of this system. Here, we determined the first cryo-electron microscopy structure of the SaCas9-sgRNA-DNA ternary complex. This structure reveals that the HNH nuclease domain is tightly bound to the cleavage site of the target DNA strand, and is in close contact with the WED and REC domains. Moreover, it captures the complete structure of the sgRNA, including the previously unresolved stem-loop 2. Based on this structure, we build a full-length model for the ternary complex in cleavage state. This model enables identification of the residues for the interactions between the HNH domain and the WED and REC domains. Moreover, we found that the stem-loop 2 of the sgRNA tightly binds to the PI and RuvC domains and may also regulate the position shift of the RuvC domain. Further mutagenesis and molecular dynamics simulations supported the idea that the interactions of the HNH domain with the WED and REC domains play an important role in the DNA cleavage. Thus, this study provides new mechanistic insights into the DNA cleavage of SaCas9 and is also useful for guiding the future engineering of SaCas9-mediated gene editing systems. Full-Length Model of SaCas9-sgRNA-DNA Complex in Cleavage State.,Du W, Zhu H, Qian J, Xue D, Zheng S, Huang Q Int J Mol Sci. 2023 Jan 7;24(2):1204. doi: 10.3390/ijms24021204. PMID:36674715[3] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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