Japan Agency for Medical Research and Development (AMED)
日本
引用
ジャーナル: Mol Cell / 年: 2022 タイトル: Structure of the type V-C CRISPR-Cas effector enzyme. 著者: Nina Kurihara / Ryoya Nakagawa / Hisato Hirano / Sae Okazaki / Atsuhiro Tomita / Kan Kobayashi / Tsukasa Kusakizako / Tomohiro Nishizawa / Keitaro Yamashita / David A Scott / Hiroshi Nishimasu / Osamu Nureki / 要旨: RNA-guided CRISPR-Cas nucleases are widely used as versatile genome-engineering tools. Recent studies identified functionally divergent type V Cas12 family enzymes. Among them, Cas12c2 binds a CRISPR ...RNA-guided CRISPR-Cas nucleases are widely used as versatile genome-engineering tools. Recent studies identified functionally divergent type V Cas12 family enzymes. Among them, Cas12c2 binds a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA) and recognizes double-stranded DNA targets with a short TN PAM. Here, we report the cryo-electron microscopy structures of the Cas12c2-guide RNA binary complex and the Cas12c2-guide RNA-target DNA ternary complex. The structures revealed that the crRNA and tracrRNA form an unexpected X-junction architecture, and that Cas12c2 recognizes a single T nucleotide in the PAM through specific hydrogen-bonding interactions with two arginine residues. Furthermore, our biochemical analyses indicated that Cas12c2 processes its precursor crRNA to a mature crRNA using the RuvC catalytic site through a unique mechanism. Collectively, our findings improve the mechanistic understanding of diverse type V CRISPR-Cas effectors.
平均露光時間: 5 sec. / 電子線照射量: 54 e/Å2 フィルム・検出器のモデル: GATAN K3 BIOQUANTUM (6k x 4k)
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解析
ソフトウェア
名称: REFMAC / バージョン: 5.8.0267 / 分類: 精密化
EMソフトウェア
ID
名称
バージョン
カテゴリ
1
RELION
3
粒子像選択
2
SerialEM
画像取得
4
CTFFIND
CTF補正
7
Coot
モデルフィッティング
9
RELION
3
初期オイラー角割当
10
RELION
3
最終オイラー角割当
11
RELION
3
分類
12
RELION
3
3次元再構成
13
REFMAC
モデル精密化
CTF補正
タイプ: PHASE FLIPPING AND AMPLITUDE CORRECTION
対称性
点対称性: C1 (非対称)
3次元再構成
解像度: 3 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 289394 / 対称性のタイプ: POINT
原子モデル構築
プロトコル: FLEXIBLE FIT / 空間: RECIPROCAL
精密化
解像度: 3→3 Å / Cor.coef. Fo:Fc: 0.908 / SU B: 13.97 / SU ML: 0.263 / ESU R: 0.428 立体化学のターゲット値: MAXIMUM LIKELIHOOD WITH PHASES 詳細: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS