- EMDB-8236: Cryo-EM structure of SpCas9-sgRNA-DNA ternary complex -
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Basic information
Entry
Database: EMDB / ID: EMD-8236
Title
Cryo-EM structure of SpCas9-sgRNA-DNA ternary complex
Map data
None
Sample
Complex: SpCas9-sgRNA-target DNA ternay complex
Function / homology
Function and homology information
maintenance of CRISPR repeat elements / 3'-5' exonuclease activity / DNA endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding Similarity search - Function
Journal: Nat Commun / Year: 2017 Title: Structural insights into DNA cleavage activation of CRISPR-Cas9 system. Authors: Cong Huai / Gan Li / Ruijie Yao / Yingyi Zhang / Mi Cao / Liangliang Kong / Chenqiang Jia / Hui Yuan / Hongyan Chen / Daru Lu / Qiang Huang / Abstract: 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.
History
Deposition
Jun 4, 2016
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Header (metadata) release
Aug 10, 2016
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Map release
Oct 25, 2017
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Update
Jan 22, 2020
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Current status
Jan 22, 2020
Processing site: PDBj / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Supramolecule #1: SpCas9-sgRNA-target DNA ternay complex
Supramolecule
Name: SpCas9-sgRNA-target DNA ternay complex / type: complex / ID: 1 / Parent: 0 Details: 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
Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 289 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 4 seconds before plunging.
Details
SpCas9 protein, sgRNA, and target DNA were cultured at 37 degree centigrade to form a complex, and then monodisperased at 18 degree centigrade overnight.
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Electron microscopy
Microscope
FEI TITAN KRIOS
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Number real images: 592 / Average electron dose: 10.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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