9WAC

Cryo-EM structure of AtCas9-sgRNA-underwound DNA (TTGA) ternary complex

Summary for 9WAC

• Entry DOI: 10.2210/pdb9wac/pdb
• EMDB information: 65811
• Descriptor: CRISPR-associated endonuclease Cas9, sgRNA, TS DNA, ... (4 entities in total)
• Functional Keywords: complex, endonuclease, immunity, rna binding protein/rna/dna, rna binding protein-rna-dna complex
• Biological source: Alicyclobacillus tolerans; More
• Total number of polymer chains: 4
• Total formula weight: 189310.74

Authors

Meng, B.,Duan, M.,Wu, L.J.,Liu, Z.J.,Zhang, Y. (deposition date: 2025-08-11, release date: 2026-07-01, Last modification date: 2026-07-22)

Primary citation

Duan, M.,Meng, B.,Zhou, L.,Wu, L.,Tong, X.,Huang, D.,Yin, H.,Liu, Z.J.,Zhang, Y.
Structural basis of AtCas9 recognition of PAM mutants in underwound DNA topology.
Nat.Struct.Mol.Biol., 33:1062-1074, 2026

PubMed Abstract: The CRISPR-Cas9 system locates targets through guide RNA pairing and recognition of a protospacer-adjacent motif (PAM). Although PAM specificity is sequence-determined, DNA topology can relax PAM requirements and enable near-PAMless cleavage by the type II-C Alicyclobacillus tengchongensis Cas9 (AtCas9). However, the structural mechanism underlying this regulation remains unknown. Here we report cryogenic-electron microscopy (cryo-EM) structures of AtCas9 bound to B-form DNA or a 340 bp underwound minicircle DNA containing wild-type or mutant PAMs. Despite PAM sequences differences, all three underwound complexes adopt an almost identical architecture distinct from the B-form DNA-bound state. On B-form DNA, AtCas9 recognizes the PAM through base-specific hydrogen bonds and steric exclusion, conferring preference for NCNNN and NRNNA (R = A/G). By contrast, underwound DNA widens the PAM major groove and promotes sequence-independent backbone contacts, explaining the near-PAMless cleavage. These findings uncover a topology-dependent mechanism of PAM recognition and establish a cryo-EM platform using underwound minicircle DNA for structural studies under native-like topological states.

PubMed: 42342973
DOI: 10.1038/s41594-026-01831-6

Experimental method

ELECTRON MICROSCOPY (2.89 Å)