9ECD

Active state of wild-type EsCas13d ternary complex with U10G mismatch

9ECD の概要

• エントリーDOI: 10.2210/pdb9ecd/pdb
• EMDBエントリー: 47907
• 分子名称: EsCas13d, crRNA, Target RNA, ... (4 entities in total)
• 機能のキーワード: cas13, crispr, hepn, rna nuclease, rna binding protein-rna complex, rna binding protein/rna
• 由来する生物種: [Eubacterium] siraeum DSM 15702; 詳細
• タンパク質・核酸の鎖数: 3
• 化学式量合計: 137220.07

構造登録者

Chou, C.W.,Finkelstein, I.J. (登録日: 2024-11-13, 公開日: 2025-11-19, 最終更新日: 2026-09-02)

主引用文献

Chou, C.W.,Sinan, S.,Kuo, H.C.,Chang, Y.C.,Arguello, C.,Sahaya, D.,Russell, R.,Finkelstein, I.J.
Structural basis for target discrimination and activation by Cas13d.
Sci Adv, 12:eaec4221-eaec4221, 2026

PubMed Abstract: CRISPR-Cas13d is increasingly used for RNA knockdowns, but off-target cleavage of near-cognate RNAs hinders its broader adoption. Here, we solve seven cryo-electron microscopy structures of wild-type Cas13d in complex with matched and mismatched targets. These structures reveal active, intermediate, and inactive states that illustrate a detailed activation mechanism. Upon target RNA binding, the CRISPR RNA undergoes marked conformational changes. The Helical-1 domain transitions from a docked state with the amino-terminal domain to an allosterically switched conformation that stabilizes the RNA duplex. Quantitative kinetics show that a single proximal mismatch preserves the binding rate constant but abolishes nuclease activity by trapping Cas13d in an inactive state. We also identify an active site loop in the higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domains that regulates substrate accessibility and can be mutated to generate both hypo- and hyperactivated variants. These findings establish the structural basis for Cas13d mismatch surveillance and provide a framework for engineering HEPN nuclease specificity and activity.

PubMed: 42616864
DOI: 10.1126/sciadv.aec4221

実験手法

ELECTRON MICROSCOPY (3.47 Å)