6PIF

V. cholerae TniQ-Cascade complex, open conformation

Summary for 6PIF

• Entry DOI: 10.2210/pdb6pif/pdb
• EMDB information: 20349
• Descriptor: Cas7, type I-F CRISPR-associated protein, cas5_8 naturally occurring fusion protein, type I-F CRISPR-associated endoribonuclease Cas6/Csy4, ... (6 entities in total)
• Functional Keywords: crispr/cas, cascade, rna binding protein, rna binding protein-rna complex, rna binding protein/rna
• Biological source: Vibrio cholerae; More
• Total number of polymer chains: 11
• Total formula weight: 421499.29

Authors

Halpin-Healy, T.,Klompe, S.,Sternberg, S.H. (deposition date: 2019-06-26, release date: 2019-10-02, Last modification date: 2024-03-20)

Primary citation

Halpin-Healy, T.S.,Klompe, S.E.,Sternberg, S.H.,Fernandez, I.S.
Structural basis of DNA targeting by a transposon-encoded CRISPR-Cas system.
Nature, 577:271-274, 2020

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. Type I CRISPR-Cas systems typically target foreign DNA for degradation via joint action of the ribonucleoprotein complex Cascade and the helicase-nuclease Cas3, but nuclease-deficient type I systems lacking Cas3 have been repurposed for RNA-guided transposition by bacterial Tn7-like transposons. 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.

PubMed: 31853065
DOI: 10.1038/s41586-019-1849-0

Experimental method

ELECTRON MICROSCOPY (3.4 Å)