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Yorodumi- PDB-6o0x: Conformational states of Cas9-sgRNA-DNA ternary complex in the pr... -
+Open data
-Basic information
Entry | Database: PDB / ID: 6o0x | ||||||||||||
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Title | Conformational states of Cas9-sgRNA-DNA ternary complex in the presence of magnesium | ||||||||||||
Components |
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Keywords | HYDROLASE/RNA/DNA / CRISPR / Cas9 / Nuclease / Genome editing / HYDROLASE / HYDROLASE-RNA-DNA 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 | ||||||||||||
Biological species | Streptococcus pyogenes (bacteria) synthetic construct (others) | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.28 Å | ||||||||||||
Authors | Zhu, X. / Clarke, R. / Puppala, A.K. / Chittori, S. / Merk, A. / Merrill, B.J. / Simonovic, M. / Subramaniam, S. | ||||||||||||
Funding support | United States, 3items
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Citation | Journal: Nat Struct Mol Biol / Year: 2019 Title: Cryo-EM structures reveal coordinated domain motions that govern DNA cleavage by Cas9. Authors: Xing Zhu / Ryan Clarke / Anupama K Puppala / Sagar Chittori / Alan Merk / Bradley J Merrill / Miljan Simonović / Sriram Subramaniam / Abstract: The RNA-guided Cas9 endonuclease from Streptococcus pyogenes is a single-turnover enzyme that displays a stable product state after double-stranded-DNA cleavage. Here, we present cryo-EM structures ...The RNA-guided Cas9 endonuclease from Streptococcus pyogenes is a single-turnover enzyme that displays a stable product state after double-stranded-DNA cleavage. Here, we present cryo-EM structures of precatalytic, postcatalytic and product states of the active Cas9-sgRNA-DNA complex in the presence of Mg. In the precatalytic state, Cas9 adopts the 'checkpoint' conformation with the HNH nuclease domain positioned far away from the DNA. Transition to the postcatalytic state involves a dramatic ~34-Å swing of the HNH domain and disorder of the REC2 recognition domain. The postcatalytic state captures the cleaved substrate bound to the catalytically competent HNH active site. In the product state, the HNH domain is disordered, REC2 returns to the precatalytic conformation, and additional interactions of REC3 and RuvC with nucleic acids are formed. The coupled domain motions and interactions between the enzyme and the RNA-DNA hybrid provide new insights into the mechanism of genome editing by Cas9. | ||||||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6o0x.cif.gz | 282 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6o0x.ent.gz | 205.5 KB | Display | PDB format |
PDBx/mmJSON format | 6o0x.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6o0x_validation.pdf.gz | 867.9 KB | Display | wwPDB validaton report |
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Full document | 6o0x_full_validation.pdf.gz | 874.6 KB | Display | |
Data in XML | 6o0x_validation.xml.gz | 34.6 KB | Display | |
Data in CIF | 6o0x_validation.cif.gz | 56.1 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/o0/6o0x ftp://data.pdbj.org/pub/pdb/validation_reports/o0/6o0x | HTTPS FTP |
-Related structure data
Related structure data | 0583MC 0584C 0585C 6o0yC 6o0zC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 158986.109 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Streptococcus pyogenes (bacteria) / Gene: cas9, csn1, SPy_1046 / Production host: Escherichia coli (E. coli) References: UniProt: Q99ZW2, Hydrolases; Acting on ester bonds |
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#2: RNA chain | Mass: 33059.699 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
#3: DNA chain | Mass: 3936.575 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
#4: DNA chain | Mass: 12452.001 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
#5: DNA chain | Mass: 8194.269 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Cas9-sgRNA-DNA ternary complex / Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES |
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Source (natural) | Organism: Streptococcus pyogenes (bacteria) |
Buffer solution | pH: 7.5 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Details: unspecified |
Vitrification | Cryogen name: ETHANE |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD |
Image recording | Electron dose: 73 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) |
-Processing
Software | Name: PHENIX / Version: 1.14_3260: / Classification: refinement |
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CTF correction | Type: NONE |
Symmetry | Point symmetry: C1 (asymmetric) |
3D reconstruction | Resolution: 3.28 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 30513 / Symmetry type: POINT |