- EMDB-8236: Cryo-EM structure of SpCas9-sgRNA-DNA ternary complex -
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基本情報
登録情報
データベース: EMDB / ID: EMD-8236
タイトル
Cryo-EM structure of SpCas9-sgRNA-DNA ternary complex
マップデータ
None
試料
複合体: SpCas9-sgRNA-target DNA ternay complex
機能・相同性
機能・相同性情報
maintenance of CRISPR repeat elements / 3'-5' exonuclease activity / DNA 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
ジャーナル: Nat Commun / 年: 2017 タイトル: Structural insights into DNA cleavage activation of CRISPR-Cas9 system. 著者: Cong Huai / Gan Li / Ruijie Yao / Yingyi Zhang / Mi Cao / Liangliang Kong / Chenqiang Jia / Hui Yuan / Hongyan Chen / Daru Lu / Qiang Huang / 要旨: CRISPR-Cas9 technology has been widely used for genome engineering. Its RNA-guided endonuclease Cas9 binds specifically to target DNA and then cleaves the two DNA strands with HNH and RuvC nuclease ...CRISPR-Cas9 technology has been widely used for genome engineering. Its RNA-guided endonuclease Cas9 binds specifically to target DNA and then cleaves the two DNA strands with HNH and RuvC nuclease domains. However, structural information regarding the DNA cleavage-activating state of two nuclease domains remains sparse. Here, we report a 5.2 Å cryo-EM structure of Cas9 in complex with sgRNA and target DNA. This structure reveals a conformational state of Cas9 in which the HNH domain is closest to the DNA cleavage site. Compared with two known HNH states, our structure shows that the HNH active site moves toward the cleavage site by about 25 and 13 Å, respectively. In combination with EM-based molecular dynamics simulations, we show that residues of the nuclease domains in our structure could form cleavage-compatible conformations with the target DNA. Together, these results strongly suggest that our cryo-EM structure resembles a DNA cleavage-activating architecture of Cas9.
名称: SpCas9-sgRNA-target DNA ternay complex / タイプ: complex / ID: 1 / 親要素: 0 詳細: S. pyogenes Cas9(D10A, H840A) in complex with a 55-mer target DNA from the fumarylacetoacetate hydrolase (FAH) gene and the corresponding 98-nucleotide sgRNA
凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 289 K / 装置: FEI VITROBOT MARK IV / 詳細: blot for 4 seconds before plunging.
詳細
SpCas9 protein, sgRNA, and target DNA were cultured at 37 degree centigrade to form a complex, and then monodisperased at 18 degree centigrade overnight.