EMD-27143

Single-particle
2.88 Å
EMD-27143 Deposition: 30/05/2022
Map released: 20/12/2023
Last modified: 23/10/2024
Overview 3D View Sample Experiment Validation Volume Browser Additional data Links
Overview 3D View Sample Experiment Validation Volume Browser Additional data Links

EMD-27143

Structure of Acidothermus cellulolyticus Cas9 ternary complex (Pre-cleavage)

EMD-27143

Single-particle
2.88 Å
EMD-27143 Deposition: 30/05/2022
Map released: 20/12/2023
Last modified: 23/10/2024
Overview 3D View Sample Experiment Validation Volume Browser Additional data Links
Sample Organism: Acidothermus cellulolyticus 11B, synthetic construct
Sample: CryoEM Structure of AceCas9 (Pre-Cleavage)
Fitted models: 8d2n (Avg. Q-score: 0.459)

Deposition Authors: Rai J, Das A, Li H
Coupled catalytic states and the role of metal coordination in Cas9.
Das A, Rai J, Roth MO , Shu Y, Medina ML , Barakat MR, Li H
(2023) Nat Catal , 6 , 969 - 977
PUBMED: 38348449
DOI: doi:10.1038/s41929-023-01031-1
ISSN: 2520-1158
Abstract:
Controlling the activity of the CRISPR-Cas9 system is essential to its safe adoption for clinical and research applications. Although the conformational dynamics of Cas9 are known to control its enzymatic activity, details of how Cas9 influences the catalytic processes at both nuclease domains remain elusive. Here we report five cryo-electron microscopy structures of the active Acidothermus cellulolyticus Cas9 complex along the reaction path at 2.2-2.9 Å resolution. We observed that a large movement in one nuclease domain, triggered by the cognate DNA, results in noticeable changes in the active site of the other domain that is required for metal coordination and catalysis. Furthermore, the conformations synchronize the reaction intermediates, enabling coupled cutting of the two DNA strands. Consistent with the roles of conformations in organizing the active sites, adjustments to the metal-coordination residues lead to altered metal specificity of A. cellulolyticus Cas9 and commonly used Streptococcus pyogenes Cas9 in cells.