5VW1

Crystal structure of SpyCas9-sgRNA-AcrIIA4 ternary complex


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.60 Å
  • R-Value Free: 0.226 
  • R-Value Work: 0.178 
  • R-Value Observed: 0.180 

wwPDB Validation   3D Report Full Report


This is version 1.4 of the entry. See complete history


Literature

Inhibition Mechanism of an Anti-CRISPR Suppressor AcrIIA4 Targeting SpyCas9.

Yang, H.Patel, D.J.

(2017) Mol Cell 67: 117-127.e5

  • DOI: https://doi.org/10.1016/j.molcel.2017.05.024
  • Primary Citation of Related Structures:  
    5VW1

  • PubMed Abstract: 

    Prokaryotic CRISPR-Cas adaptive immune systems utilize sequence-specific RNA-guided endonucleases to defend against infection by viruses, bacteriophages, and mobile elements, while these foreign genetic elements evolve diverse anti-CRISPR proteins to overcome the CRISPR-Cas-mediated defense of the host. Recently, AcrIIA2 and AcrIIA4, encoded by Listeria monocytogene prophages, were shown to block the endonuclease activity of type II-A Streptococcus pyogene Cas9 (SpyCas9). We now report the crystal structure of AcrIIA4 in complex with single-guide RNA-bound SpyCas9, thereby establishing that AcrIIA4 preferentially targets critical residues essential for PAM duplex recognition, as well as blocks target DNA access to key catalytic residues lining the RuvC pocket. These structural insights, validated by biochemical assays on key mutants, demonstrate that AcrIIA4 competitively occupies both PAM-interacting and non-target DNA strand cleavage catalytic pockets. Our studies provide insights into anti-CRISPR-mediated suppression mechanisms for inactivating SpyCas9, thereby broadening the applicability of CRISPR-Cas regulatory tools for genome editing.


  • Organizational Affiliation

    Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Electronic address: yangh3@mskcc.org.


Macromolecules

Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
CRISPR-associated endonuclease Cas9/Csn11,370Streptococcus pyogenes serotype M1Mutation(s): 2 
Gene Names: cas9csn1SPy_1046
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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  • Reference Sequence
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Entity ID: 3
MoleculeChains Sequence LengthOrganismDetailsImage
anti-CRISPR protein AcrIIA4C [auth B]89Listeria monocytogenes M7Mutation(s): 0 
Gene Names: LMM7_0114
UniProt
Find proteins for A0A0E0UT28 (Listeria monocytogenes serotype 4a (strain M7))
Explore A0A0E0UT28 
Go to UniProtKB:  A0A0E0UT28
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupA0A0E0UT28
Sequence Annotations
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  • Reference Sequence
Find similar nucleic acids by:  (by identity cutoff)  |  3D Structure
Entity ID: 2
MoleculeChains LengthOrganismImage
sgRNAB [auth C]85Listeria monocytogenes
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.60 Å
  • R-Value Free: 0.226 
  • R-Value Work: 0.178 
  • R-Value Observed: 0.180 
  • Space Group: P 2 21 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 72.194α = 90
b = 101.242β = 90
c = 303.563γ = 90
Software Package:
Software NamePurpose
PHENIXrefinement
HKL-2000data scaling
PDB_EXTRACTdata extraction
HKL-2000data reduction
PHENIXphasing

Structure Validation

View Full Validation Report



Entry History & Funding Information

Deposition Data


Funding OrganizationLocationGrant Number
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)United StatesGM104962
Memorial Sloan Kettering Cancer Center Core GrantUnited StatesP30CA008748

Revision History  (Full details and data files)

  • Version 1.0: 2017-06-28
    Type: Initial release
  • Version 1.1: 2017-07-19
    Changes: Database references
  • Version 1.2: 2017-09-20
    Changes: Author supporting evidence
  • Version 1.3: 2020-01-01
    Changes: Author supporting evidence
  • Version 1.4: 2023-10-04
    Changes: Data collection, Database references, Refinement description