SNAP output for PDB entry 5u0a [SNAP web server]

PDB-id

5u0a; SNAP-derived features in text and JSON formats; DNAproDB

Class

immune system

Method

cryo-EM (3.3 Å)

Summary

Crispr RNA-guided surveillance complex

Reference

Xiao Y, Luo M, Hayes RP, Kim J, Ng S, Ding F, Liao M, Ke A (2017): "Structure Basis for Directional R-loop Formation and Substrate Handover Mechanisms in Type I CRISPR-Cas System." Cell, 170, 48-60.e11. doi: 10.1016/j.cell.2017.06.012.

Abstract

Type I CRISPR systems feature a sequential dsDNA target searching and degradation process, by crRNA-displaying Cascade and nuclease-helicase fusion enzyme Cas3, respectively. Here we present two cryo-EM snapshots of the Thermobifida fusca type I-E Cascade: (1) unwinding 11 bp of dsDNA at the seed-sequence region to scout for sequence complementarity, and (2) further unwinding of the entire protospacer to form a full R-loop. These structures provide the much-needed temporal and spatial resolution to resolve key mechanistic steps leading to Cas3 recruitment. In the early steps, PAM recognition causes severe DNA bending, leading to spontaneous DNA unwinding to form a seed-bubble. The full R-loop formation triggers conformational changes in Cascade, licensing Cas3 to bind. The same process also generates a bulge in the non-target DNA strand, enabling its handover to Cas3 for cleavage. The combination of both negative and positive checkpoints ensures stringent yet efficient target degradation in type I CRISPR-Cas systems.