4UN5

Crystal structure of Cas9 bound to PAM-containing DNA target containing mismatches at positions 1-3


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.40 Å
  • R-Value Free: 0.246 
  • R-Value Work: 0.215 
  • R-Value Observed: 0.216 

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This is version 1.3 of the entry. See complete history


Literature

Structural Basis of Pam-Dependent Target DNA Recognition by the Cas9 Endonuclease

Anders, C.Niewoehner, O.Duerst, A.Jinek, M.

(2014) Nature 513: 569

  • DOI: https://doi.org/10.1038/nature13579
  • Primary Citation of Related Structures:  
    4UN3, 4UN4, 4UN5

  • PubMed 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.


  • Organizational Affiliation

    Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.


Macromolecules

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Entity ID: 2
MoleculeChains Sequence LengthOrganismDetailsImage
CRISPR-ASSOCIATED ENDONUCLEASE CAS9/CSN11,372Streptococcus pyogenesMutation(s): 2 
EC: 3.1
UniProt
Find proteins for Q99ZW2 (Streptococcus pyogenes serotype M1)
Explore Q99ZW2 
Go to UniProtKB:  Q99ZW2
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ99ZW2
Sequence Annotations
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Entity ID: 1
MoleculeChains LengthOrganismImage
SGRNA83synthetic construct
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Entity ID: 3
MoleculeChains LengthOrganismImage
TARGET DNA STRAND PROXIMAL FRAGMENT11synthetic construct
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Entity ID: 4
MoleculeChains LengthOrganismImage
NON-TARGET DNA STRAND11synthetic construct
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Entity ID: 5
MoleculeChains LengthOrganismImage
TARGET DNA STRAND DISTAL FRAGMENT17synthetic construct
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.40 Å
  • R-Value Free: 0.246 
  • R-Value Work: 0.215 
  • R-Value Observed: 0.216 
  • Space Group: C 1 2 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 180.182α = 90
b = 68.072β = 111.37
c = 190.261γ = 90
Software Package:
Software NamePurpose
PHENIXrefinement
XDSdata reduction
SCALAdata scaling
PHASERphasing

Structure Validation

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Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2014-07-23
    Type: Initial release
  • Version 1.1: 2014-08-13
    Changes: Database references
  • Version 1.2: 2014-10-01
    Changes: Database references
  • Version 1.3: 2024-01-10
    Changes: Data collection, Database references, Derived calculations, Other, Refinement description