8HHL

Cryo-EM structure of the Cas12m2-crRNA-target DNA full R-loop complex

Summary for 8HHL

• Entry DOI: 10.2210/pdb8hhl/pdb
• EMDB information: 34803
• Descriptor: crRNA (56-MER), TS (36-MER), NTS (36-MER), ... (6 entities in total)
• Functional Keywords: crispr-cas, rna binding protein-dna complex, rna binding protein
• Biological source: Mycolicibacterium mucogenicum; More
• Total number of polymer chains: 4
• Total formula weight: 107029.48

Authors

Omura, N.S.,Nakagawa, R.,Wu, Y.W.,Sudfeld, C.,Warren, V.R.,Hirano, H.,Kusakizako, T.,Kise, Y.,Lebbink, H.G.J.,Itoh, Y.,Oost, V.D.J.,Nureki, O. (deposition date: 2022-11-16, release date: 2023-04-12, Last modification date: 2023-08-30)

Primary citation

Omura, S.N.,Nakagawa, R.,Sudfeld, C.,Villegas Warren, R.,Wu, W.Y.,Hirano, H.,Laffeber, C.,Kusakizako, T.,Kise, Y.,Lebbink, J.H.G.,Itoh, Y.,van der Oost, J.,Nureki, O.
Mechanistic and evolutionary insights into a type V-M CRISPR-Cas effector enzyme.
Nat.Struct.Mol.Biol., 30:1172-1182, 2023

PubMed Abstract: RNA-guided type V CRISPR-Cas12 effectors provide adaptive immunity against mobile genetic elements (MGEs) in bacteria and archaea. Among diverse Cas12 enzymes, the recently identified Cas12m2 (CRISPR-Cas type V-M) is highly compact and has a unique RuvC active site. Although the non-canonical RuvC triad does not permit dsDNA cleavage, Cas12m2 still protects against invading MGEs through transcriptional silencing by strong DNA binding. However, the molecular mechanism of RNA-guided genome inactivation by Cas12m2 remains unknown. Here we report cryo-electron microscopy structures of two states of Cas12m2-CRISPR RNA (crRNA)-target DNA ternary complexes and the Cas12m2-crRNA binary complex, revealing structural dynamics during crRNA-target DNA heteroduplex formation. The structures indicate that the non-target DNA strand is tightly bound to a unique arginine-rich cluster in the recognition (REC) domains and the non-canonical active site in the RuvC domain, ensuring strong DNA-binding affinity of Cas12m2. Furthermore, a structural comparison of Cas12m2 with TnpB, a putative ancestor of Cas12 enzymes, suggests that the interaction of the characteristic coiled-coil REC2 insertion with the protospacer-adjacent motif-distal region of the heteroduplex is crucial for Cas12m2 to engage in adaptive immunity. Collectively, our findings improve mechanistic understanding of diverse type V CRISPR-Cas effectors and provide insights into the evolution of TnpB to Cas12 enzymes.

PubMed: 37460897
DOI: 10.1038/s41594-023-01042-3

Experimental method

ELECTRON MICROSCOPY (2.87 Å)