- EMDB-32104: Cryo-EM structure of SaCas9-sgRNA-DNA ternary complex -
+
データを開く
IDまたはキーワード:
読み込み中...
-
基本情報
登録情報
データベース: EMDB / ID: EMD-32104
タイトル
Cryo-EM structure of SaCas9-sgRNA-DNA ternary complex
マップデータ
試料
複合体: SaCas9-sgRNA-target DNA ternary complex
複合体: SaCas9
タンパク質・ペプチド: SaCas9
複合体: sgRNA-target DNA
RNA: single-guide RNA (sgRNA)
DNA: Target DNA strand(TS)
DNA: Non-target DNA strand (NTS)
機能・相同性
機能・相同性情報
maintenance of CRISPR repeat elements / endonuclease activity / defense response to virus / 加水分解酵素; エステル加水分解酵素 / DNA binding / RNA binding / metal ion binding 類似検索 - 分子機能
National Natural Science Foundation of China (NSFC)
31971377
中国
National Natural Science Foundation of China (NSFC)
31671386
中国
引用
ジャーナル: Int J Mol Sci / 年: 2023 タイトル: Full-Length Model of SaCas9-sgRNA-DNA Complex in Cleavage State. 著者: Wenhao Du / Haixia Zhu / Jiaqiang Qian / Dongmei Xue / Sen Zheng / Qiang Huang / 要旨: Cas9 (SaCas9) is a widely used genome editing tool. Understanding its molecular mechanisms of DNA cleavage could effectively guide the engineering optimization of this system. Here, we determined ... Cas9 (SaCas9) is a widely used genome editing tool. Understanding its molecular mechanisms of DNA cleavage could effectively guide the engineering optimization of this system. Here, we determined the first cryo-electron microscopy structure of the SaCas9-sgRNA-DNA ternary complex. This structure reveals that the HNH nuclease domain is tightly bound to the cleavage site of the target DNA strand, and is in close contact with the WED and REC domains. Moreover, it captures the complete structure of the sgRNA, including the previously unresolved stem-loop 2. Based on this structure, we build a full-length model for the ternary complex in cleavage state. This model enables identification of the residues for the interactions between the HNH domain and the WED and REC domains. Moreover, we found that the stem-loop 2 of the sgRNA tightly binds to the PI and RuvC domains and may also regulate the position shift of the RuvC domain. Further mutagenesis and molecular dynamics simulations supported the idea that the interactions of the HNH domain with the WED and REC domains play an important role in the DNA cleavage. Thus, this study provides new mechanistic insights into the DNA cleavage of SaCas9 and is also useful for guiding the future engineering of SaCas9-mediated gene editing systems.
選択した数: 3872337 詳細: We used the program PARSED developed in our previous study to pick the complex particles from the micrographs(Yao R, et al. Bioinformatics. 2020, 36:1252-1259).