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Yorodumi- PDB-8d2l: Structure of Acidothermus cellulolyticus Cas9 ternary complex (Cl... -
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-Basic information
Entry | Database: PDB / ID: 8d2l | |||||||||
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Title | Structure of Acidothermus cellulolyticus Cas9 ternary complex (Cleavage Intermediate 1) | |||||||||
Components |
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Keywords | RNA BINDING PROTEIN/DNA/RNA / Cas9 / AcCas9 / Crispr / RNA BINDING PROTEIN / RNA BINDING PROTEIN-DNA-RNA complex | |||||||||
Function / homology | Function and homology information defense response to virus / endonuclease activity / DNA binding / RNA binding / zinc ion binding Similarity search - Function | |||||||||
Biological species | Acidothermus cellulolyticus 11B (bacteria) synthetic construct (others) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.21 Å | |||||||||
Authors | Rai, J. / Das, A. / Li, H. | |||||||||
Funding support | United States, 2items
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Citation | Journal: Nat Catal / Year: 2023 Title: Coupled catalytic states and the role of metal coordination in Cas9. Authors: Anuska Das / Jay Rai / Mitchell O Roth / Yuerong Shu / Megan L Medina / Mackenzie R Barakat / Hong Li / Abstract: Controlling the activity of the CRISPR-Cas9 system is essential to its safe adoption for clinical and research applications. Although the conformational dynamics of Cas9 are known to control its ...Controlling the activity of the CRISPR-Cas9 system is essential to its safe adoption for clinical and research applications. Although the conformational dynamics of Cas9 are known to control its enzymatic activity, details of how Cas9 influences the catalytic processes at both nuclease domains remain elusive. Here we report five cryo-electron microscopy structures of the active Cas9 complex along the reaction path at 2.2-2.9 Å resolution. We observed that a large movement in one nuclease domain, triggered by the cognate DNA, results in noticeable changes in the active site of the other domain that is required for metal coordination and catalysis. Furthermore, the conformations synchronize the reaction intermediates, enabling coupled cutting of the two DNA strands. Consistent with the roles of conformations in organizing the active sites, adjustments to the metal-coordination residues lead to altered metal specificity of Cas9 and commonly used Cas9 in cells. | |||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8d2l.cif.gz | 373 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8d2l.ent.gz | 245.9 KB | Display | PDB format |
PDBx/mmJSON format | 8d2l.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8d2l_validation.pdf.gz | 1.4 MB | Display | wwPDB validaton report |
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Full document | 8d2l_full_validation.pdf.gz | 1.4 MB | Display | |
Data in XML | 8d2l_validation.xml.gz | 42.2 KB | Display | |
Data in CIF | 8d2l_validation.cif.gz | 66.8 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/d2/8d2l ftp://data.pdbj.org/pub/pdb/validation_reports/d2/8d2l | HTTPS FTP |
-Related structure data
Related structure data | 27142MC 8d2kC 8d2nC 8d2oC 8d2pC 8d2qC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-DNA target strand (5'- ... , 2 types, 2 molecules XT
#3: DNA chain | Mass: 4021.632 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
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#4: DNA chain | Mass: 7216.654 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
-DNA non-target strand (5'- ... , 2 types, 2 molecules DY
#5: DNA chain | Mass: 3919.588 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
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#6: DNA chain | Mass: 910.662 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
-Protein / RNA chain , 2 types, 2 molecules AB
#1: Protein | Mass: 127498.094 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Acidothermus cellulolyticus 11B (bacteria) Strain: ATCC 43068 / DSM 8971 / 11B / Gene: Acel_1951 / Production host: Escherichia coli (E. coli) / References: UniProt: A0LWB3 |
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#2: RNA chain | Mass: 34390.289 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Acidothermus cellulolyticus 11B (bacteria) Strain: ATCC 43068 / DSM 8971 / 11B / Production host: Escherichia coli (E. coli) |
-Non-polymers , 2 types, 351 molecules
#7: Chemical | ChemComp-MG / #8: Water | ChemComp-HOH / | |
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-Details
Has ligand of interest | N |
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-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 |
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Source (natural) | Organism: Acidothermus cellulolyticus 11B (bacteria) | ||||||||||||||||||||||||
Source (recombinant) | Organism: Escherichia coli (E. coli) | ||||||||||||||||||||||||
Buffer solution | pH: 7.5 | ||||||||||||||||||||||||
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % |
-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 / Nominal defocus max: 2200 nm / Nominal defocus min: 1000 nm |
Image recording | Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
EM imaging optics | Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV |
-Processing
Software |
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EM software | Name: RELION / Version: 4 / Category: final Euler assignment | ||||||||||||||||||||||||
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
3D reconstruction | Resolution: 2.21 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 196600 / Symmetry type: POINT | ||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||
Displacement parameters | Biso mean: 28.47 Å2 | ||||||||||||||||||||||||
Refine LS restraints |
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