6PIJ

Target DNA-bound V. cholerae TniQ-Cascade complex, closed conformation


Experimental Data Snapshot

  • Method: ELECTRON MICROSCOPY
  • Resolution: 2.90 Å
  • Aggregation State: PARTICLE 
  • Reconstruction Method: SINGLE PARTICLE 

wwPDB Validation   3D Report Full Report


This is version 1.3 of the entry. See complete history


Literature

Structural basis of DNA targeting by a transposon-encoded CRISPR-Cas system.

Halpin-Healy, T.S.Klompe, S.E.Sternberg, S.H.Fernandez, I.S.

(2020) Nature 577: 271-274

  • DOI: https://doi.org/10.1038/s41586-019-1849-0
  • Primary Citation of Related Structures:  
    6PIF, 6PIG, 6PIJ

  • PubMed Abstract: 

    Bacteria use adaptive immune systems encoded by CRISPR and Cas genes to maintain genomic integrity when challenged by pathogens and mobile genetic elements 1-3 . Type I CRISPR-Cas systems typically target foreign DNA for degradation via joint action of the ribonucleoprotein complex Cascade and the helicase-nuclease Cas3 4,5 , but nuclease-deficient type I systems lacking Cas3 have been repurposed for RNA-guided transposition by bacterial Tn7-like transposons 6,7 . How CRISPR- and transposon-associated machineries collaborate during DNA targeting and insertion remains unknown. Here we describe structures of a TniQ-Cascade complex encoded by the Vibrio cholerae Tn6677 transposon using cryo-electron microscopy, revealing the mechanistic basis of this functional coupling. The cryo-electron microscopy maps enabled de novo modelling and refinement of the transposition protein TniQ, which binds to the Cascade complex as a dimer in a head-to-tail configuration, at the interface formed by Cas6 and Cas7 near the 3' end of the CRISPR RNA (crRNA). The natural Cas8-Cas5 fusion protein binds the 5' crRNA handle and contacts the TniQ dimer via a flexible insertion domain. A target DNA-bound structure reveals critical interactions necessary for protospacer-adjacent motif recognition and R-loop formation. This work lays the foundation for a structural understanding of how DNA targeting by TniQ-Cascade leads to downstream recruitment of additional transposase proteins, and will guide protein engineering efforts to leverage this system for programmable DNA insertions in genome-engineering applications.


  • Organizational Affiliation

    Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA.


Macromolecules

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Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
cas7 type I-F CRISPR-associated protein Csy3
A, B, C, D, E
A, B, C, D, E, F
351Vibrio choleraeMutation(s): 0 
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Entity ID: 2
MoleculeChains Sequence LengthOrganismDetailsImage
cas5_8 naturally occurring fusion protein511Vibrio choleraeMutation(s): 0 
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Entity ID: 3
MoleculeChains Sequence LengthOrganismDetailsImage
type I-F CRISPR-associated endoribonuclease Cas6/Csy4197Vibrio choleraeMutation(s): 0 
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Entity ID: 4
MoleculeChains Sequence LengthOrganismDetailsImage
TniQ monomer I358Vibrio choleraeMutation(s): 0 
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Entity ID: 5
MoleculeChains Sequence LengthOrganismDetailsImage
TniQ monomer 2369Vibrio choleraeMutation(s): 0 
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Entity ID: 6
MoleculeChains LengthOrganismImage
guide RNAK [auth 1]60Vibrio cholerae
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Entity ID: 7
MoleculeChains LengthOrganismImage
Targeting strand ssDNAL [auth 2]27Vibrio cholerae
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Entity ID: 8
MoleculeChains LengthOrganismImage
Non-targeting strand ssDNAM [auth 3]9Vibrio cholerae
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Experimental Data & Validation

Experimental Data

  • Method: ELECTRON MICROSCOPY
  • Resolution: 2.90 Å
  • Aggregation State: PARTICLE 
  • Reconstruction Method: SINGLE PARTICLE 

Structure Validation

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Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2019-10-02
    Type: Initial release
  • Version 1.1: 2020-01-22
    Changes: Database references
  • Version 1.2: 2020-06-24
    Changes: Structure summary
  • Version 1.3: 2024-03-20
    Changes: Data collection, Database references