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Yorodumi- PDB-4oge: Crystal structure of the Type II-C Cas9 enzyme from Actinomyces n... -
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-Basic information
Entry | Database: PDB / ID: 4oge | ||||||
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Title | Crystal structure of the Type II-C Cas9 enzyme from Actinomyces naeslundii | ||||||
Components | HNH endonuclease domain protein | ||||||
Keywords | HYDROLASE / CRISPR-Cas / Cas9 / HNH / RuvC / RNA-guided DNA endonuclease / cytoplasmic | ||||||
Function / homology | Function and homology information endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / zinc ion binding Similarity search - Function | ||||||
Biological species | Actinomyces naeslundii (bacteria) | ||||||
Method | X-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 2.201 Å | ||||||
Authors | Jiang, F. / Ma, E. / Lin, S. / Doudna, J.A. | ||||||
Citation | Journal: Science / Year: 2014 Title: Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Authors: Martin Jinek / Fuguo Jiang / David W Taylor / Samuel H Sternberg / Emine Kaya / Enbo Ma / Carolin Anders / Michael Hauer / Kaihong Zhou / Steven Lin / Matias Kaplan / Anthony T Iavarone / ...Authors: Martin Jinek / Fuguo Jiang / David W Taylor / Samuel H Sternberg / Emine Kaya / Enbo Ma / Carolin Anders / Michael Hauer / Kaihong Zhou / Steven Lin / Matias Kaplan / Anthony T Iavarone / Emmanuelle Charpentier / Eva Nogales / Jennifer A Doudna / Abstract: Type II CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA ...Type II CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA during an adaptive bacterial immune response. Cas9 has been harnessed as a powerful tool for genome editing and gene regulation in many eukaryotic organisms. We report 2.6 and 2.2 angstrom resolution crystal structures of two major Cas9 enzyme subtypes, revealing the structural core shared by all Cas9 family members. The architectures of Cas9 enzymes define nucleic acid binding clefts, and single-particle electron microscopy reconstructions show that the two structural lobes harboring these clefts undergo guide RNA-induced reorientation to form a central channel where DNA substrates are bound. The observation that extensive structural rearrangements occur before target DNA duplex binding implicates guide RNA loading as a key step in Cas9 activation. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 4oge.cif.gz | 398.9 KB | Display | PDBx/mmCIF format |
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PDB format | pdb4oge.ent.gz | 321.8 KB | Display | PDB format |
PDBx/mmJSON format | 4oge.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 4oge_validation.pdf.gz | 446.5 KB | Display | wwPDB validaton report |
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Full document | 4oge_full_validation.pdf.gz | 463.1 KB | Display | |
Data in XML | 4oge_validation.xml.gz | 38.6 KB | Display | |
Data in CIF | 4oge_validation.cif.gz | 55.2 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/og/4oge ftp://data.pdbj.org/pub/pdb/validation_reports/og/4oge | HTTPS FTP |
-Related structure data
Related structure data | 5858C 5859C 5860C 4cmpC 4cmqC 4ogcC 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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Unit cell |
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-Components
#1: Protein | Mass: 123958.703 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Details: pET-based expression vector with an N-terminal His10-tag followed by Maltose-Binding Protein (MBP) and a TEV protease cleavage site. Source: (gene. exp.) Actinomyces naeslundii (bacteria) / Strain: Howell 279 / Gene: HMPREF1129_2620 / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta (DE3) / References: UniProt: J3F2B0 | ||||
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#2: Chemical | ChemComp-ZN / | ||||
#3: Chemical | #4: Chemical | #5: Water | ChemComp-HOH / | |
-Experimental details
-Experiment
Experiment | Method: X-RAY DIFFRACTION / Number of used crystals: 1 |
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-Sample preparation
Crystal | Density Matthews: 3.25 Å3/Da / Density % sol: 62.11 % |
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Crystal grow | Temperature: 293 K / Method: vapor diffusion, hanging drop / pH: 6.5 Details: 10% (w/v) PEG 8000, 0.25 M calcium acetate, 50 mM magnesium acetate and 5 mM spermidine, pH 6.5, VAPOR DIFFUSION, HANGING DROP, temperature 293K |
-Data collection
Diffraction | Mean temperature: 100 K |
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Diffraction source | Source: SYNCHROTRON / Site: ALS / Beamline: 8.3.1 / Wavelength: 1.116 Å |
Detector | Type: ADSC QUANTUM 315r / Detector: CCD / Date: Sep 21, 2013 |
Radiation | Monochromator: Double flat crystal, Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray |
Radiation wavelength | Wavelength: 1.116 Å / Relative weight: 1 |
Reflection | Resolution: 2.2→80.2 Å / Num. all: 676466 / Num. obs: 78425 / % possible obs: 98 % / Observed criterion σ(F): 2 / Observed criterion σ(I): 2.4 / Redundancy: 8.6 % / Biso Wilson estimate: 36.341 Å2 |
Reflection shell | Resolution: 2.2→2.32 Å / Redundancy: 5.8 % / Num. unique all: 10087 / % possible all: 86.8 |
-Processing
Software |
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Refinement | Method to determine structure: SAD / Resolution: 2.201→68.692 Å / SU ML: 0.26 / σ(F): 1.36 / σ(I): 2.4 / Phase error: 23.47 / Stereochemistry target values: ML
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Solvent computation | Shrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refinement step | Cycle: LAST / Resolution: 2.201→68.692 Å
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Refine LS restraints |
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LS refinement shell |
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Refinement TLS params. | Method: refined / Refine-ID: X-RAY DIFFRACTION
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Refinement TLS group |
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