8H4U

Cryo-EM structure of a riboendonuclease

8H4U の概要

• エントリーDOI: 10.2210/pdb8h4u/pdb
• EMDBエントリー: 34484
• 分子名称: CRISPR-associated endonuclease Cas9 (1 entity in total)
• 機能のキーワード: riboendonuclease, rna
• 由来する生物種: Thermoclostridium caenicola
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 143582.64

構造登録者

Li, Z.,Wang, F. (登録日: 2022-10-11, 公開日: 2023-08-30, 最終更新日: 2024-03-13)

主引用文献

Wang, F.,Zhang, C.,Xu, H.,Zeng, W.,Ma, L.,Li, Z.
Structural Basis for the Ribonuclease Activity of a Thermostable CRISPR-Cas13a from Thermoclostridium caenicola.
J.Mol.Biol., 435:168197-168197, 2023

PubMed Abstract: The RNA-targeting type VI CRISPR-Cas effector complexes are widely used in biotechnology applications such as gene knockdown, RNA editing, and molecular diagnostics. Compared with Cas13a from mesophilic organisms, a newly discovered Cas13a from thermophilic bacteria Thermoclostridium caenicola (TccCas13a) shows low sequence similarity, high thermostability, and lacks pre-crRNA processing activity. The thermostability of TccCas13a has been harnessed to make a sensitive and robust tool for nucleic acid detection. Here we present the structures of TccCas13a-crRNA binary complex at 2.8 Å, and TccCas13a at 3.5 Å. Although TccCas13a shares a similarly bilobed architecture with other mesophilic organism-derived Cas13a proteins, TccCas13a displayed distinct structure features. Specifically, it holds a long crRNA 5'-flank, forming extensive polar contacts with Helical-1 and HEPN2 domains. The detailed analysis of the interaction between crRNA 5'-flank and TccCas13a suggested lack of suitable nucleophile to attack the 2'-OH of crRNA 5'-flank may explain why TccCas13a fails to cleave pre-crRNA. The stem-loop segment of crRNA spacer toggles between double-stranded and single-stranded conformational states, suggesting a potential safeguard mechanism for target recognition. Superimposition of the structures of TccCas13a and TccCas13a-crRNA revealed several conformational changes required for crRNA loading, including dramatic movement of Helical-2 domain. Collectively, these structural insights expand our understanding into type VI CRISPR-Cas effectors, and would facilitate the development of TccCas13a-based applications.

PubMed: 37442412
DOI: 10.1016/j.jmb.2023.168197

実験手法

ELECTRON MICROSCOPY (3.5 Å)