5fw2: Difference between revisions
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The | ==Crystal structure of SpCas9 variant EQR bound to sgRNA and TGAG PAM target DNA== | ||
<StructureSection load='5fw2' size='340' side='right'caption='[[5fw2]], [[Resolution|resolution]] 2.68Å' scene=''> | |||
== Structural highlights == | |||
<table><tr><td colspan='2'>[[5fw2]] is a 4 chain structure with sequence from [https://en.wikipedia.org/wiki/Streptococcus_pyogenes Streptococcus pyogenes] 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=5FW2 OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=5FW2 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]] 2.676Å</td></tr> | |||
<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=K:POTASSIUM+ION'>K</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=5fw2 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=5fw2 OCA], [https://pdbe.org/5fw2 PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=5fw2 RCSB], [https://www.ebi.ac.uk/pdbsum/5fw2 PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=5fw2 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 == | |||
The RNA-guided endonuclease Cas9 from Streptococcus pyogenes (SpCas9) forms the core of a powerful genome editing technology. DNA cleavage by SpCas9 is dependent on the presence of a 5'-NGG-3' protospacer adjacent motif (PAM) in the target DNA, restricting the choice of targetable sequences. To address this limitation, artificial SpCas9 variants with altered PAM specificities have recently been developed. Here we report crystal structures of the VQR, EQR, and VRER SpCas9 variants bound to target DNAs containing their preferred PAM sequences. The structures reveal that the non-canonical PAMs are recognized by an induced fit mechanism. Besides mediating sequence-specific base recognition, the amino acid substitutions introduced in the SpCas9 variants facilitate conformational remodeling of the PAM region of the bound DNA. Guided by the structural data, we engineered a SpCas9 variant that specifically recognizes NAAG PAMs. Taken together, these studies inform further development of Cas9-based genome editing tools. | |||
Structural Plasticity of PAM Recognition by Engineered Variants of the RNA-Guided Endonuclease Cas9.,Anders C, Bargsten K, Jinek M Mol Cell. 2016 Mar 17;61(6):895-902. doi: 10.1016/j.molcel.2016.02.020. PMID:26990992<ref>PMID:26990992</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
[[Category: | </div> | ||
[[Category: | <div class="pdbe-citations 5fw2" style="background-color:#fffaf0;"></div> | ||
[[Category: Bargsten | |||
[[Category: | ==See Also== | ||
*[[CRISPR-Cas|CRISPR-Cas]] | |||
*[[CRISPR-Cas Part II|CRISPR-Cas Part II]] | |||
*[[CRISPR-Cas9|CRISPR-Cas9]] | |||
*[[Endonuclease 3D structures|Endonuclease 3D structures]] | |||
== References == | |||
<references/> | |||
__TOC__ | |||
</StructureSection> | |||
[[Category: Large Structures]] | |||
[[Category: Streptococcus pyogenes]] | |||
[[Category: Synthetic construct]] | |||
[[Category: Anders C]] | |||
[[Category: Bargsten K]] | |||
[[Category: Jinek M]] | |||