+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-8236 | |||||||||
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Title | Cryo-EM structure of SpCas9-sgRNA-DNA ternary complex | |||||||||
Map data | None | |||||||||
Sample |
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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 | |||||||||
Biological species | Streptococcus pyogenes (bacteria) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 5.2 Å | |||||||||
Authors | Li G / Huang Q / Huai C | |||||||||
Funding support | China, 2 items
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Citation | 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_8236.map.gz | 9.6 MB | EMDB map data format | |
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Header (meta data) | emd-8236-v30.xml emd-8236.xml | 13.6 KB 13.6 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_8236_fsc.xml | 5 KB | Display | FSC data file |
Images | emd_8236.png | 111.7 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-8236 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-8236 | HTTPS FTP |
-Validation report
Summary document | emd_8236_validation.pdf.gz | 359.4 KB | Display | EMDB validaton report |
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Full document | emd_8236_full_validation.pdf.gz | 358.9 KB | Display | |
Data in XML | emd_8236_validation.xml.gz | 7.8 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-8236 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-8236 | HTTPS FTP |
-Related structure data
Related structure data | 5y36MC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_8236.map.gz / Format: CCP4 / Size: 10.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | None | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.3 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : SpCas9-sgRNA-target DNA ternay complex
Entire | Name: SpCas9-sgRNA-target DNA ternay complex |
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Components |
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-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 |
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Source (natural) | Organism: Streptococcus pyogenes (bacteria) |
Molecular weight | Theoretical: 225 KDa |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.45 mg/mL | |||||||||||||||
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Buffer | pH: 7.5 Component:
Details: 20mM Tris-Cl (pH 7.5), 100mM KCl, 5mM MgCl2, 1mM DTT | |||||||||||||||
Grid | 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. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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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 |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.3 µm / Nominal magnification: 18000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |