8SFP

WT CRISPR-Cas12a with the target strand in the RuvC active site.

8SFP の概要

• エントリーDOI: 10.2210/pdb8sfp/pdb
• EMDBエントリー: 40447
• 分子名称: CRISPR-associated endonuclease Cas12a, RNA (39-MER), DNA (40-MER), ... (4 entities in total)
• 機能のキーワード: crispr, r-loop, endonuclease, dna binding protein, dna binding protein-dna-rna complex, dna binding protein/dna/rna
• 由来する生物種: Acidaminococcus sp. BV3L6; 詳細
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 201567.37

構造登録者

Strohkendl, I.,Taylor, D.W. (登録日: 2023-04-11, 公開日: 2024-07-03, 最終更新日: 2025-01-22)

主引用文献

Strohkendl, I.,Saha, A.,Moy, C.,Nguyen, A.H.,Ahsan, M.,Russell, R.,Palermo, G.,Taylor, D.W.
Cas12a domain flexibility guides R-loop formation and forces RuvC resetting.
Mol.Cell, 84:2717-2731.e6, 2024

PubMed Abstract: The specific nature of CRISPR-Cas12a makes it a desirable RNA-guided endonuclease for biotechnology and therapeutic applications. To understand how R-loop formation within the compact Cas12a enables target recognition and nuclease activation, we used cryo-electron microscopy to capture wild-type Acidaminococcus sp. Cas12a R-loop intermediates and DNA delivery into the RuvC active site. Stages of Cas12a R-loop formation-starting from a 5-bp seed-are marked by distinct REC domain arrangements. Dramatic domain flexibility limits contacts until nearly complete R-loop formation, when the non-target strand is pulled across the RuvC nuclease and coordinated domain docking promotes efficient cleavage. Next, substantial domain movements enable target strand repositioning into the RuvC active site. Between cleavage events, the RuvC lid conformationally resets to occlude the active site, requiring re-activation. These snapshots build a structural model depicting Cas12a DNA targeting that rationalizes observed specificity and highlights mechanistic comparisons to other class 2 effectors.

PubMed: 38955179
DOI: 10.1016/j.molcel.2024.06.007

実験手法

ELECTRON MICROSCOPY (3.8 Å)