6PIJ

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

6PIJ の概要

• エントリーDOI: 10.2210/pdb6pij/pdb
• EMDBエントリー: 20351
• 分子名称: cas7 type I-F CRISPR-associated protein Csy3, cas5_8 naturally occurring fusion protein, type I-F CRISPR-associated endoribonuclease Cas6/Csy4, ... (8 entities in total)
• 機能のキーワード: crispr/cas, cascade, rna binding protein, rna binding protein-rna-dna complex, rna binding protein/rna/dna
• 由来する生物種: Vibrio cholerae; 詳細
• タンパク質・核酸の鎖数: 13
• 化学式量合計: 431522.41

構造登録者

Halpin-Healy, T.,Klompe, S.,Sternberg, S.H. (登録日: 2019-06-26, 公開日: 2019-10-02, 最終更新日: 2024-03-20)

主引用文献

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

実験手法

ELECTRON MICROSCOPY (2.9 Å)