SNAP annotations: PDB entry 9g9e

PDB-id

9g9e; DSSR-derived features in text and JSON formats; DNAproDB

Class

RNA binding protein

Method

cryo-EM (2.87 Å)

Summary

Cryoem structure of enterococcus italicus csm-crrna complex bound to ampnpp

Reference

Jungfer K, Moravcik S, Garcia-Doval C, Knorlein A, Hall J, Jinek M (2025): "Mechanistic determinants and dynamics of cA6 synthesis in type III CRISPR-Cas effector complexes." Nucleic Acids Res., 53. doi: 10.1093/nar/gkae1277.

Abstract

Type III clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems (type III CRISPR-Cas systems) use guide RNAs to recognize RNA transcripts of foreign genetic elements, which triggers the generation of cyclic oligoadenylate (cOA) second messengers by the Cas10 subunit of the type III effector complex. In turn, cOAs bind and activate ancillary effector proteins to reinforce the host immune response. Type III systems utilize distinct cOAs, including cyclic tri- (cA3), tetra- (cA4) and hexa-adenylates (cA6). However, the molecular mechanisms dictating cOA product identity are poorly understood. Here we used cryoelectron microscopy to visualize the mechanism of cA6 biosynthesis by the Csm effector complex from Enterococcus italicus (EiCsm). We show that EiCsm synthesizes oligoadenylate nucleotides in 3'-5' direction using a set of conserved binding sites in the Cas10 Palm domains to determine the size of the nascent oligoadenylate chain. Our data also reveal that conformational dynamics induced by target RNA binding results in allosteric activation of Cas10 to trigger oligoadenylate synthesis. Mutations of a key structural element in Cas10 perturb cOA synthesis to favor cA3 and cA4 formation. Together, these results provide comprehensive insights into the dynamics of cOA synthesis in type III CRISPR-Cas systems and reveal key determinants of second messenger product selectivity, thereby illuminating potential avenues for their engineering.