+Open data
-Basic information
Entry | Database: PDB / ID: 5vzl | ||||||
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Title | cryo-EM structure of the Cas9-sgRNA-AcrIIA4 anti-CRISPR complex | ||||||
Components |
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Keywords | Immune System/RNA / anti-CRISPR / Cas9 / CRISPR / gene editing / on-target / off-target / cryo-EM / Immune System-RNA complex | ||||||
Function / homology | Function and homology information symbiont-mediated suppression of host CRISPR-cas system / 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 M1 GAS (bacteria) unidentified phage (virus) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å | ||||||
Authors | Jiang, F. / Liu, J.J. / Nogales, E. / Doudna, J.A. | ||||||
Funding support | United States, 1items
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Citation | Journal: Sci Adv / Year: 2017 Title: Disabling Cas9 by an anti-CRISPR DNA mimic. Authors: Jiyung Shin / Fuguo Jiang / Jun-Jie Liu / Nicolas L Bray / Benjamin J Rauch / Seung Hyun Baik / Eva Nogales / Joseph Bondy-Denomy / Jacob E Corn / Jennifer A Doudna / Abstract: CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 gene editing technology is derived from a microbial adaptive immune system, where bacteriophages are often the intended target. ...CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 gene editing technology is derived from a microbial adaptive immune system, where bacteriophages are often the intended target. Natural inhibitors of CRISPR-Cas9 enable phages to evade immunity and show promise in controlling Cas9-mediated gene editing in human cells. However, the mechanism of CRISPR-Cas9 inhibition is not known, and the potential applications for Cas9 inhibitor proteins in mammalian cells have not been fully established. We show that the anti-CRISPR protein AcrIIA4 binds only to assembled Cas9-single-guide RNA (sgRNA) complexes and not to Cas9 protein alone. A 3.9 Å resolution cryo-electron microscopy structure of the Cas9-sgRNA-AcrIIA4 complex revealed that the surface of AcrIIA4 is highly acidic and binds with a 1:1 stoichiometry to a region of Cas9 that normally engages the DNA protospacer adjacent motif. Consistent with this binding mode, order-of-addition experiments showed that AcrIIA4 interferes with DNA recognition but has no effect on preformed Cas9-sgRNA-DNA complexes. Timed delivery of AcrIIA4 into human cells as either protein or expression plasmid allows on-target Cas9-mediated gene editing while reducing off-target edits. These results provide a mechanistic understanding of AcrIIA4 function and demonstrate that inhibitors can modulate the extent and outcomes of Cas9-mediated gene editing. | ||||||
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 | 5vzl.cif.gz | 326.5 KB | Display | PDBx/mmCIF format |
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PDB format | pdb5vzl.ent.gz | 251.5 KB | Display | PDB format |
PDBx/mmJSON format | 5vzl.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 5vzl_validation.pdf.gz | 792.4 KB | Display | wwPDB validaton report |
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Full document | 5vzl_full_validation.pdf.gz | 814.9 KB | Display | |
Data in XML | 5vzl_validation.xml.gz | 44.7 KB | Display | |
Data in CIF | 5vzl_validation.cif.gz | 70.3 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/vz/5vzl ftp://data.pdbj.org/pub/pdb/validation_reports/vz/5vzl | HTTPS FTP |
-Related structure data
Related structure data | 8749MC 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: RNA chain | Mass: 38292.645 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Streptococcus pyogenes M1 GAS (bacteria) Gene: csn1, cas9, ERS445054_00848 / Production host: Escherichia coli (E. coli) |
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#2: Protein | Mass: 158772.891 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Streptococcus pyogenes M1 GAS (bacteria) Gene: csn1, cas9, ERS445054_00848 / Production host: Escherichia coli (E. coli) References: UniProt: A0A0C6FZC2, UniProt: Q99ZW2*PLUS, Hydrolases; Acting on ester bonds |
#3: Protein | Mass: 10182.073 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) unidentified phage (virus) / Production host: Escherichia coli (E. coli) / References: UniProt: A0A2D0TCG7*PLUS |
-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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Molecular weight | Experimental value: NO | ||||||||||||||||||||||||
Source (natural) |
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Source (recombinant) | Organism: Escherichia coli (E. coli) | ||||||||||||||||||||||||
Buffer solution | pH: 8 | ||||||||||||||||||||||||
Specimen | Conc.: 1.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: spyCas9, sgRNA and AcrIIA4 complex | ||||||||||||||||||||||||
Specimen support | Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-2/2 | ||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K Details: blot for 4.5 seconds with force of 12 before plunging |
-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: 3000 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 900 nm / Calibrated defocus max: 4000 nm / Cs: 0.01 mm / C2 aperture diameter: 100 µm |
Image recording | Electron dose: 46 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) |
-Processing
Software | Name: PHENIX / Version: 1.11.1_2575: / Classification: refinement | ||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 185000 / Symmetry type: POINT | ||||||||||||||||||||||||
Atomic model building |
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Refine LS restraints |
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