spacer
spacer

PDBsum entry 5fw3

Go to PDB code: 
protein dna_rna metals links
Hydrolase/DNA/RNA PDB id
5fw3
Jmol PyMol
Contents
Protein chain
1322 a.a.
DNA/RNA
Metals
__K ×11
_MG ×2
Waters ×152
PDB id:
5fw3
Name: Hydrolase/DNA/RNA
Title: Crystal structure of spcas9 variant vrer bound to sgrna and tgcg pam target DNA
Structure: Crispr-associated endonuclease cas9/csn1. Chain: b. Synonym: cas9 vrer variant, spcas9. Engineered: yes. Mutation: yes. Sgrna. Chain: a. Target DNA strand. Chain: c.
Source: Streptococcus pyogenes. Organism_taxid: 1314. Strain: m1. Expressed in: escherichia coli. Expression_system_taxid: 469008. Expression_system_variant: rosetta 2. Synthetic: yes. Synthetic construct. Organism_taxid: 32630.
Resolution:
2.70Å     R-factor:   0.215     R-free:   0.241
Authors: C.Anders,K.Bargsten,M.Jinek
Key ref: C.Anders et al. (2016). Structural Plasticity of PAM Recognition by Engineered Variants of the RNA-Guided Endonuclease Cas9. Mol Cell, 61, 895-902. PubMed id: 26990992 DOI: 10.1016/j.molcel.2016.02.020
Date:
11-Feb-16     Release date:   15-Jun-16    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chain
Pfam   ArchSchema ?
Q99ZW2  (Q99ZW2_STRP1) -  CRISPR-associated endonuclease Cas9/Csn1
Seq:
Struc:
 
Seq:
Struc:
 
Seq:
Struc:
1368 a.a.
1322 a.a.*
Key:    PfamA domain  Secondary structure
* PDB and UniProt seqs differ at 6 residue positions (black crosses)

 Enzyme reactions 
   Enzyme class: E.C.3.1.-.-
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     nucleic acid phosphodiester bond hydrolysis   3 terms 
  Biochemical function     hydrolase activity     9 terms  

 

 
DOI no: 10.1016/j.molcel.2016.02.020 Mol Cell 61:895-902 (2016)
PubMed id: 26990992  
 
 
Structural Plasticity of PAM Recognition by Engineered Variants of the RNA-Guided Endonuclease Cas9.
C.Anders, K.Bargsten, M.Jinek.
 
  ABSTRACT  
 
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.
 

 

spacer

spacer