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PDBsum entry 5b2t

Go to PDB code: 
protein dna_rna ligands metals links
Hydrolase/RNA/DNA PDB id
5b2t
Jmol PyMol
Contents
Protein chain
1326 a.a.
DNA/RNA
Ligands
EDO ×5
ACT
Metals
__K ×10
_MG ×3
Waters ×608
PDB id:
5b2t
Name: Hydrolase/RNA/DNA
Title: Crystal structure of the streptococcus pyogenes cas9 vrer va complex with sgrna and target DNA (tgcg pam)
Structure: Guide RNA. Chain: a. Engineered: yes. Crispr-associated endonuclease cas9. Chain: b. Synonym: spycas9. Engineered: yes. Mutation: yes. Target DNA.
Source: Synthetic: yes. Streptococcus pyogenes. Organism_taxid: 1314. Other_details: in vitro transcription. Streptococcus pyogenes serotype m1. Organism_taxid: 301447. Strain: serotype m1. Gene: cas9, csn1, spy_1046. Expressed in: escherichia coli.
Resolution:
2.20Å     R-factor:   0.205     R-free:   0.223
Authors: S.Hirano,H.Nishimasu,R.Ishitani,O.Nureki
Key ref: S.Hirano et al. (2016). Structural Basis for the Altered PAM Specificities of Engineered CRISPR-Cas9. Mol Cell, 61, 886-894. PubMed id: 26990991 DOI: 10.1016/j.molcel.2016.02.018
Date:
02-Feb-16     Release date:   23-Mar-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.
1326 a.a.*
Key:    PfamA domain  Secondary structure
* PDB and UniProt seqs differ at 8 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.2016.02.018 Mol Cell 61:886-894 (2016)
PubMed id: 26990991  
 
 
Structural Basis for the Altered PAM Specificities of Engineered CRISPR-Cas9.
S.Hirano, H.Nishimasu, R.Ishitani, O.Nureki.
 
  ABSTRACT  
 
The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets bearing a PAM (protospacer adjacent motif) and complementarity to the guide RNA. A recent study showed that, whereas wild-type Streptococcus pyogenes Cas9 (SpCas9) recognizes the 5'-NGG-3' PAM, the engineered VQR, EQR, and VRER SpCas9 variants recognize the 5'-NGA-3', 5'-NGAG-3', and 5'-NGCG-3' PAMs, respectively, thus expanding the targetable sequences in Cas9-mediated genome editing applications. Here, we present the high-resolution crystal structures of the three SpCas9 variants in complexes with a single-guide RNA and its altered PAM-containing, partially double-stranded DNA targets. A structural comparison of the three SpCas9 variants with wild-type SpCas9 revealed that the multiple mutations synergistically induce an unexpected displacement in the phosphodiester backbone of the PAM duplex, thereby allowing the SpCas9 variants to directly recognize the altered PAM nucleotides. OurĀ findings explain the altered PAM specificities of the SpCas9 variants and establish a framework for further rational engineering of CRISPR-Cas9.
 

 

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