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PDBsum entry 4un3

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protein dna_rna metals links
Hydrolase/DNA/RNA PDB id
4un3
Jmol PyMol
Contents
Protein chain
1306 a.a.
DNA/RNA
Metals
_MG ×8
Waters ×405
PDB id:
4un3
Name: Hydrolase/DNA/RNA
Title: Crystal structure of cas9 bound to pam-containing DNA target
Structure: Crispr-associated endonuclease cas9/csn1. Chain: b. Synonym: cas9. 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:
2.59Å     R-factor:   0.218     R-free:   0.252
Authors: C.Anders,O.Niewoehner,A.Duerst,M.Jinek
Key ref: C.Anders et al. (2014). Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature, 513, 569-573. PubMed id: 25079318 DOI: 10.1038/nature13579
Date:
25-May-14     Release date:   23-Jul-14    
PROCHECK
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 Headers
 References

Protein chain
Pfam   ArchSchema ?
Q99ZW2  (Q99ZW2_STRP1) -  CRISPR-associated endonuclease Cas9/Csn1
Seq:
Struc:
 
Seq:
Struc:
 
Seq:
Struc:
1368 a.a.
1306 a.a.*
Key:    PfamA domain  Secondary structure
* PDB and UniProt seqs differ at 1 residue position (black cross)

 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: 10.1038/nature13579 Nature 513:569-573 (2014)
PubMed id: 25079318  
 
 
Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease.
C.Anders, O.Niewoehner, A.Duerst, M.Jinek.
 
  ABSTRACT  
 
The CRISPR-associated protein Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA bearing sequences complementary to a 20-nucleotide segment in the guide RNA. Cas9 has emerged as a versatile molecular tool for genome editing and gene expression control. RNA-guided DNA recognition and cleavage strictly require the presence of a protospacer adjacent motif (PAM) in the target DNA. Here we report a crystal structure of Streptococcus pyogenes Cas9 in complex with a single-molecule guide RNA and a target DNA containing a canonical 5'-NGG-3' PAM. The structure reveals that the PAM motif resides in a base-paired DNA duplex. The non-complementary strand GG dinucleotide is read out via major-groove interactions with conserved arginine residues from the carboxy-terminal domain of Cas9. Interactions with the minor groove of the PAM duplex and the phosphodiester group at the +1 position in the target DNA strand contribute to local strand separation immediately upstream of the PAM. These observations suggest a mechanism for PAM-dependent target DNA melting and RNA-DNA hybrid formation. Furthermore, this study establishes a framework for the rational engineering of Cas9 enzymes with novel PAM specificities.
 

 

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