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- PDB-5f9r: Crystal structure of catalytically-active Streptococcus pyogenes ... -
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Open data
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Basic information
Entry | Database: PDB / ID: 5f9r | ||||||
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Title | Crystal structure of catalytically-active Streptococcus pyogenes CRISPR-Cas9 in complex with single-guided RNA and double-stranded DNA primed for target DNA cleavage | ||||||
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![]() | HYDROLASE/DNA/RNA / CRISPR / Cas9 / R-loop / genome engineering / HYDROLASE-DNA-RNA complex | ||||||
Function / homology | ![]() 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 | ![]() ![]() ![]() | ||||||
Method | ![]() ![]() ![]() | ||||||
![]() | Jiang, F. / Doudna, J.A. | ||||||
Funding support | ![]()
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![]() | ![]() Title: Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage. Authors: Fuguo Jiang / David W Taylor / Janice S Chen / Jack E Kornfeld / Kaihong Zhou / Aubri J Thompson / Eva Nogales / Jennifer A Doudna / ![]() Abstract: Bacterial adaptive immunity and genome engineering involving the CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) protein Cas9 begin with RNA-guided DNA unwinding ...Bacterial adaptive immunity and genome engineering involving the CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) protein Cas9 begin with RNA-guided DNA unwinding to form an RNA-DNA hybrid and a displaced DNA strand inside the protein. The role of this R-loop structure in positioning each DNA strand for cleavage by the two Cas9 nuclease domains is unknown. We determine molecular structures of the catalytically active Streptococcus pyogenes Cas9 R-loop that show the displaced DNA strand located near the RuvC nuclease domain active site. These protein-DNA interactions, in turn, position the HNH nuclease domain adjacent to the target DNA strand cleavage site in a conformation essential for concerted DNA cutting. Cas9 bends the DNA helix by 30°, providing the structural distortion needed for R-loop formation. | ||||||
History |
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 371.2 KB | Display | ![]() |
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PDB format | ![]() | 282.2 KB | Display | ![]() |
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-Validation report
Summary document | ![]() | 483.6 KB | Display | ![]() |
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Full document | ![]() | 526.6 KB | Display | |
Data in XML | ![]() | 55.2 KB | Display | |
Data in CIF | ![]() | 76.2 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
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Links
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Assembly
Deposited unit | ![]()
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Unit cell |
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Components
#1: Protein | Mass: 158699.844 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() Gene: cas9, csn1, SPy_1046 / Production host: ![]() ![]() References: UniProt: Q99ZW2, Hydrolases; Acting on ester bonds |
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#2: RNA chain | Mass: 38292.645 Da / Num. of mol.: 1 / Source method: obtained synthetically Details: RNA was prepared by in vitro transcription with T7 RNA polymerase Source: (synth.) ![]() |
#3: DNA chain | Mass: 9085.841 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() |
#4: DNA chain | Mass: 9362.040 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() |
#5: Chemical |
-Experimental details
-Experiment
Experiment | Method: ![]() |
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Sample preparation
Crystal | Density Matthews: 4.2 Å3/Da / Density % sol: 70.75 % |
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Crystal grow | Temperature: 293 K / Method: vapor diffusion, hanging drop / pH: 8 Details: 1.8 M ammonium sulfate, 100 mM Tris-HCl pH8.0 and 10 mM EDTA |
-Data collection
Diffraction | Mean temperature: 100 K | ||||||||||||
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Diffraction source | Source: ![]() ![]() ![]() | ||||||||||||
Detector | Type: ADSC QUANTUM 315r / Detector: CCD / Date: Aug 25, 2015 | ||||||||||||
Radiation | Protocol: MAD / Monochromatic (M) / Laue (L): M / Scattering type: x-ray | ||||||||||||
Radiation wavelength |
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Reflection | Resolution: 3.4→119 Å / Num. obs: 48447 / % possible obs: 99 % / Redundancy: 3.8 % / Rsym value: 0.056 / Net I/σ(I): 6.7 | ||||||||||||
Reflection shell | Resolution: 3.4→3.6 Å / Redundancy: 3.6 % / Rmerge(I) obs: 0.52 / Mean I/σ(I) obs: 1.9 / % possible all: 99.7 |
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Processing
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Refinement | Method to determine structure: ![]()
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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: 3.4→69.725 Å
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Refine LS restraints |
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LS refinement shell |
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