9ASH

Cryo-EM structure of the active Lactococcus lactis Csm bound to target in post-cleavage stage

Summary for 9ASH

• Entry DOI: 10.2210/pdb9ash/pdb
• EMDB information: 43814
• Descriptor: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A), MAGNESIUM ION, CRISPR-associated protein Csm4, ... (11 entities in total)
• Functional Keywords: type iii-a crispr-cas, csm, cyclic oligoadenylate synthesis, rna binding protein-rna complex, rna binding protein/rna
• Biological source: Lactococcus lactis subsp. lactis; More
• Total number of polymer chains: 13
• Total formula weight: 333638.46

Authors

Wang, B.,Goswami, H.N.,Li, H. (deposition date: 2024-02-25, release date: 2024-08-14, Last modification date: 2024-10-30)

Primary citation

Goswami, H.N.,Ahmadizadeh, F.,Wang, B.,Addo-Yobo, D.,Zhao, Y.,Whittington, A.C.,He, H.,Terns, M.P.,Li, H.
Molecular basis for cA6 synthesis by a type III-A CRISPR-Cas enzyme and its conversion to cA4 production.
Nucleic Acids Res., 52:10619-10629, 2024

PubMed Abstract: The type III-A (Csm) CRISPR-Cas systems are multi-subunit and multipronged prokaryotic enzymes in guarding the hosts against viral invaders. Beyond cleaving activator RNA transcripts, Csm confers two additional activities: shredding single-stranded DNA and synthesizing cyclic oligoadenylates (cOAs) by the Cas10 subunit. Known Cas10 enzymes exhibit a fascinating diversity in cOA production. Three major forms-cA3, cA4 and cA6have been identified, each with the potential to trigger unique downstream effects. Whereas the mechanism for cOA-dependent activation is well characterized, the molecular basis for synthesizing different cOA isoforms remains unclear. Here, we present structural characterization of a cA6-producing Csm complex during its activation by an activator RNA. Analysis of the captured intermediates of cA6 synthesis suggests a 3'-to-5' nucleotidyl transferring process. Three primary adenine binding sites can be identified along the chain elongation path, including a unique tyrosine-threonine dyad found only in the cA6-producing Cas10. Consistently, disrupting the tyrosine-threonine dyad specifically impaired cA6 production while promoting cA4 production. These findings suggest that Cas10 utilizes a unique enzymatic mechanism for forming the phosphodiester bond and has evolved distinct strategies to regulate the cOA chain length.

PubMed: 38989619
DOI: 10.1093/nar/gkae603

Experimental method

ELECTRON MICROSCOPY (2.58 Å)