6WC0

Crystal structure of AceCas9 bound with guide RNA and DNA with 5'-NNNTC-3' PAM

6WC0 の概要

• エントリーDOI: 10.2210/pdb6wc0/pdb
• 関連するPDBエントリー: 6WBR
• 分子名称: CRISPR-associated endonuclease, Csn1 family, sgRNA (95-MER), DNA (30-MER), ... (4 entities in total)
• 機能のキーワード: dna endonuclease, crispr-cas9, hnh, ruvc, rna binding protein, rna binding protein-rna-dna complex, rna binding protein/rna/dna
• 由来する生物種: Acidothermus cellulolyticus (strain ATCC 43068 / 11B); 詳細
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 152255.02

構造登録者

Li, H.,Das, A. (登録日: 2020-03-28, 公開日: 2020-11-18, 最終更新日: 2023-10-18)

主引用文献

Das, A.,Hand, T.H.,Smith, C.L.,Wickline, E.,Zawrotny, M.,Li, H.
The molecular basis for recognition of 5'-NNNCC-3' PAM and its methylation state by Acidothermus cellulolyticus Cas9.
Nat Commun, 11:6346-6346, 2020

PubMed Abstract: Acidothermus cellulolyticus CRISPR-Cas9 (AceCas9) is a thermophilic Type II-C enzyme that has potential genome editing applications in extreme environments. It cleaves DNA with a 5'-NNNCC-3' Protospacer Adjacent Motif (PAM) and is sensitive to its methylation status. To understand the molecular basis for the high specificity of AceCas9 for its PAM, we determined two crystal structures of AceCas9 lacking its HNH domain (AceCas9-ΔHNH) bound with a single guide RNA and DNA substrates, one with the correct and the other with an incorrect PAM. Three residues, Glu1044, Arg1088, Arg1091, form an intricate hydrogen bond network with the first cytosine and the two opposing guanine nucleotides to confer specificity. Methylation of the first but not the second cytosine base abolishes AceCas9 activity, consistent with the observed PAM recognition pattern. The high sensitivity of AceCas9 to the modified cytosine makes it a potential device for detecting epigenomic changes in genomes.

PubMed: 33311465
DOI: 10.1038/s41467-020-20204-1

実験手法

X-RAY DIFFRACTION (3.61 Å)