This old version of Proteopedia is provided for student assignments while the new version is undergoing repairs. Content and edits done in this old version of Proteopedia after March 1, 2026 will eventually be lost when it is retired in about June of 2026.

Apply for new accounts at the new Proteopedia. Your logins will work in both the old and new versions.

5fw1

From Proteopedia

(Difference between revisions)

Jump to: navigation, search

Revision as of 07:36, 23 October 2019

Crystal structure of SpyCas9 variant VQR bound to sgRNA and TGAG PAM target DNA

Structural highlights

5fw1 is a 4 chain structure with sequence from "micrococcus_scarlatinae"_klein_1884 "micrococcus scarlatinae" klein 1884. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance.
Ligands:	,
Related:	5fw2, 5fw3
Resources:	FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT

Function

[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.^[1] ^[2]

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^[3]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

References

↑ Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, Eckert MR, Vogel J, Charpentier E. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature. 2011 Mar 31;471(7340):602-7. doi: 10.1038/nature09886. PMID:21455174 doi:http://dx.doi.org/10.1038/nature09886
↑ Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012 Aug 17;337(6096):816-21. doi: 10.1126/science.1225829. Epub 2012, Jun 28. PMID:22745249 doi:http://dx.doi.org/10.1126/science.1225829
↑ Anders C, Bargsten K, Jinek M. Structural Plasticity of PAM Recognition by Engineered Variants of the RNA-Guided Endonuclease Cas9. Mol Cell. 2016 Mar 17;61(6):895-902. doi: 10.1016/j.molcel.2016.02.020. PMID:26990992 doi:http://dx.doi.org/10.1016/j.molcel.2016.02.020

1 Structural highlights
2 Function
3 Publication Abstract from PubMed
4 See Also
5 References

Proteopedia Page Contributors and Editors (what is this?)

OCA

Retrieved from "http://52.214.119.220/wiki/index.php/5fw1"

@@ Line 1: / Line 1: @@
 ==Crystal structure of SpyCas9 variant VQR bound to sgRNA and TGAG PAM target DNA==
-<StructureSection load='5fw1' size='340' side='right' caption='[[5fw1]], [[Resolution|resolution]] 2.50&Aring;' scene=''>
+<StructureSection load='5fw1' size='340' side='right'caption='[[5fw1]], [[Resolution|resolution]] 2.50&Aring;' scene=''>
 == Structural highlights ==
 <table><tr><td colspan='2'>[[5fw1]] is a 4 chain structure with sequence from [http://en.wikipedia.org/wiki/"micrococcus_scarlatinae"_klein_1884 "micrococcus scarlatinae" klein 1884]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=5FW1 OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=5FW1 FirstGlance]. <br>
@@ Line 19: / Line 19: @@
 </div>
 <div class="pdbe-citations 5fw1" style="background-color:#fffaf0;"></div>
+==See Also==
+*[[Endonuclease 3D structures|Endonuclease 3D structures]]
 == References ==
 <references/>
@@ Line 24: / Line 27: @@
 </StructureSection>
 [[Category: Micrococcus scarlatinae klein 1884]]
+[[Category: Large Structures]]
 [[Category: Anders, C]]
 [[Category: Bargsten, K]]

5fw1

From Proteopedia

Revision as of 07:36, 23 October 2019

Crystal structure of SpyCas9 variant VQR bound to sgRNA and TGAG PAM target DNA

Structural highlights

Function

Publication Abstract from PubMed

See Also

References

Contents

Proteopedia Page Contributors and Editors (what is this?)

Views

Personal tools

Navigation

Search

Toolbox