6GTC

Transition state structure of Cpf1(Cas12a) I1 conformation

6GTC の概要

• エントリーDOI: 10.2210/pdb6gtc/pdb
• EMDBエントリー: 0061
• 分子名称: CRISPR-associated endonuclease Cas12a, RNA (28-MER), DNA (5'-D(P*TP*GP*AP*CP*TP*TP*CP*TP*CP*TP*AP*AP*CP*AP*AP*GP*CP*TP*CP*G)-3'), ... (4 entities in total)
• 機能のキーワード: genome editing crispr ribonucleoprotein complex, hydrolase
• 由来する生物種: Francisella tularensis subsp. novicida U112; 詳細
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 203280.74

構造登録者

Mesa, P.,Montoya, G. (登録日: 2018-06-18, 公開日: 2018-12-19, 最終更新日: 2024-05-15)

主引用文献

Stella, S.,Mesa, P.,Thomsen, J.,Paul, B.,Alcon, P.,Jensen, S.B.,Saligram, B.,Moses, M.E.,Hatzakis, N.S.,Montoya, G.
Conformational Activation Promotes CRISPR-Cas12a Catalysis and Resetting of the Endonuclease Activity.
Cell, 175:1856-1871.e21, 2018

PubMed 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.

PubMed: 30503205
DOI: 10.1016/j.cell.2018.10.045

実験手法

ELECTRON MICROSCOPY (3.91 Å)