PDBsum entry 6pij

RNA binding protein/RNA/DNA

PDB id: 6pij

Contents

Protein chains

351 a.a.

310 a.a.

511 a.a.

197 a.a.

358 a.a.

369 a.a.

DNA/RNA

PDB id:

6pij

Name:

RNA binding protein/RNA/DNA

Title:

Target DNA-bound v. Cholerae tniq-cascade complex, closed conformation

Structure:

Cas7 type i-f crispr-associated protein csy3. Chain: a, b, c, d, e, f. Engineered: yes. Cas5_8 naturally occurring fusion protein. Chain: g. Engineered: yes. Type i-f crispr-associated endoribonuclease cas6/csy4. Chain: h. Engineered: yes.

Source:

Vibrio cholerae. Organism_taxid: 666. Expressed in: escherichia coli. Expression_system_taxid: 562. Synthetic: yes. Organism_taxid: 666

Authors:

T.Halpin-Healy,S.Klompe,S.H.Sternberg

Key ref:

T.S.Halpin-Healy et al. (2020). Structural basis of DNA targeting by a transposon-encoded CRISPR-Cas system. Nature, 577, 271-274. PubMed id: 31853065 DOI: 10.1038/s41586-019-1849-0

Date:

26-Jun-19 Release date: 02-Oct-19

Protein chains

No UniProt id for this chain

Struc: 351 a.a.

Protein chain

No UniProt id for this chain

Struc: 310 a.a.

Protein chain

No UniProt id for this chain

Struc: 511 a.a.

Protein chain

No UniProt id for this chain

Struc: 197 a.a.

Protein chain

No UniProt id for this chain

Struc: 358 a.a.

Protein chain

No UniProt id for this chain

Struc: 369 a.a.

DNA/RNA chains

C-U-G-A-U-A-A-C-U-U-A-C-A-G-G-A-C-G-C-U-U-U-G-G-C-U-U-C-A-U-U-G-C-U-U-U-U-C-A- 60 bases

A-T-G-A-A-G-C-C-A-A-G-G-C-G-T-C-C-T-G-T-A-A-G-G-C-G-G 27 bases

C-C-G-C-C-T-T-A-C 9 bases

DOI no: 10.1038/s41586-019-1849-0

Nature 577:271-274 (2020)

PubMed id: 31853065

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

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

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.