EMD-0062

Single-particle
4.24 Å
EMD-0062 Deposition: 18/06/2018
Map released: 19/12/2018
Last modified: 15/05/2024
Overview 3D View Sample Experiment Validation Volume Browser Additional data Links
Overview 3D View Sample Experiment Validation Volume Browser Additional data Links

EMD-0062

Transient state structure of CRISPR-Cpf1 (Cas12a). I2 conformation

EMD-0062

Single-particle
4.24 Å
EMD-0062 Deposition: 18/06/2018
Map released: 19/12/2018
Last modified: 15/05/2024
Overview 3D View Sample Experiment Validation Volume Browser Additional data Links
Sample Organism: Francisella tularensis subsp. novicida U112
Sample: TRansition state complex I2 conformation
Fitted models: 6gtd (Avg. Q-score: 0.384)

Deposition Authors: Montoya G , Mesa P
Conformational Activation Promotes CRISPR-Cas12a Catalysis and Resetting of the Endonuclease Activity.
Stella S , Mesa P, Thomsen J , Paul B , Alcon P , Jensen SB , Saligram B, Moses ME , Hatzakis NS , Montoya G
(2018) Cell , 175 , 1856 - 1871.e21
PUBMED: 30503205
DOI: doi:10.1016/j.cell.2018.10.045
ISSN: 1097-4172
Abstract:
Cas12a, also known as Cpf1, is a type V-A CRISPR-Cas RNA-guided endonuclease that is used for genome editing based on its ability to generate specific dsDNA breaks. Here, we show cryo-EM structures of intermediates of the cleavage reaction, thus visualizing three protein regions that sense the crRNA-DNA hybrid assembly triggering the catalytic activation of Cas12a. Single-molecule FRET provides the thermodynamics and kinetics of the conformational activation leading to phosphodiester bond hydrolysis. These findings illustrate why Cas12a cuts its target DNA and unleashes unspecific cleavage activity, degrading ssDNA molecules after activation. In addition, we show that other crRNAs are able to displace the R-loop inside the protein after target DNA cleavage, terminating indiscriminate ssDNA degradation. We propose a model whereby the conformational activation of the enzyme results in indiscriminate ssDNA cleavage. The displacement of the R-loop by a new crRNA molecule will reset Cas12a specificity, targeting new DNAs.