+Open data
-Basic information
Entry | Database: PDB / ID: 6vqw | ||||||||||||||||||
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Title | Type I-F CRISPR-Csy complex with its inhibitor AcrF8 | ||||||||||||||||||
Components |
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Keywords | RNA BINDING PROTEIN/RNA/INHIBITOR / CRISPR / Type I-F / Csy / AcrF8 / anti-CRISPR / RNA BINDING PROTEIN-RNA-INHIBITOR complex | ||||||||||||||||||
Function / homology | Function and homology information maintenance of CRISPR repeat elements / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / RNA binding Similarity search - Function | ||||||||||||||||||
Biological species | Pseudomonas aeruginosa (bacteria) | ||||||||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.42 Å | ||||||||||||||||||
Authors | Zhang, K. / Li, S. / Pintilie, G. / Zhu, Y. / Huang, Z. / Chiu, W. | ||||||||||||||||||
Funding support | United States, China, 5items
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Citation | Journal: Proc Natl Acad Sci U S A / Year: 2020 Title: Inhibition mechanisms of AcrF9, AcrF8, and AcrF6 against type I-F CRISPR-Cas complex revealed by cryo-EM. Authors: Kaiming Zhang / Shuo Wang / Shanshan Li / Yuwei Zhu / Grigore D Pintilie / Tung-Chung Mou / Michael F Schmid / Zhiwei Huang / Wah Chiu / Abstract: Prokaryotes and viruses have fought a long battle against each other. Prokaryotes use CRISPR-Cas-mediated adaptive immunity, while conversely, viruses evolve multiple anti-CRISPR (Acr) proteins to ...Prokaryotes and viruses have fought a long battle against each other. Prokaryotes use CRISPR-Cas-mediated adaptive immunity, while conversely, viruses evolve multiple anti-CRISPR (Acr) proteins to defeat these CRISPR-Cas systems. The type I-F CRISPR-Cas system in requires the crRNA-guided surveillance complex (Csy complex) to recognize the invading DNA. Although some Acr proteins against the Csy complex have been reported, other relevant Acr proteins still need studies to understand their mechanisms. Here, we obtain three structures of previously unresolved Acr proteins (AcrF9, AcrF8, and AcrF6) bound to the Csy complex using electron cryo-microscopy (cryo-EM), with resolution at 2.57 Å, 3.42 Å, and 3.15 Å, respectively. The 2.57-Å structure reveals fine details for each molecular component within the Csy complex as well as the direct and water-mediated interactions between proteins and CRISPR RNA (crRNA). Our structures also show unambiguously how these Acr proteins bind differently to the Csy complex. AcrF9 binds to key DNA-binding sites on the Csy spiral backbone. AcrF6 binds at the junction between Cas7.6f and Cas8f, which is critical for DNA duplex splitting. AcrF8 binds to a distinct position on the Csy spiral backbone and forms interactions with crRNA, which has not been seen in other Acr proteins against the Csy complex. Our structure-guided mutagenesis and biochemistry experiments further support the anti-CRISPR mechanisms of these Acr proteins. Our findings support the convergent consequence of inhibiting degradation of invading DNA by these Acr proteins, albeit with different modes of interactions with the type I-F CRISPR-Cas system. | ||||||||||||||||||
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 | 6vqw.cif.gz | 471 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6vqw.ent.gz | 375.8 KB | Display | PDB format |
PDBx/mmJSON format | 6vqw.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6vqw_validation.pdf.gz | 954.2 KB | Display | wwPDB validaton report |
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Full document | 6vqw_full_validation.pdf.gz | 981.9 KB | Display | |
Data in XML | 6vqw_validation.xml.gz | 69.7 KB | Display | |
Data in CIF | 6vqw_validation.cif.gz | 110.2 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/vq/6vqw ftp://data.pdbj.org/pub/pdb/validation_reports/vq/6vqw | HTTPS FTP |
-Related structure data
Related structure data | 21359MC 6vqvC 6vqxC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-Protein , 3 types, 3 molecules CJA
#1: Protein | Mass: 36244.074 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) Gene: csy2, ALP65_00953, EQH76_13810, FCG96_17775, PACL_0128 Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) References: UniProt: B3G161, UniProt: Q02MM0*PLUS |
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#3: Protein | Mass: 21427.504 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Gene: cas6f, csy4, PA14_33300 Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) References: UniProt: Q02MM2, Hydrolases; Acting on ester bonds |
#4: Protein | Mass: 10206.353 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) |
-CRISPR-associated protein ... , 2 types, 7 molecules DEFGHIB
#2: Protein | Mass: 39778.594 Da / Num. of mol.: 6 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Gene: EQH76_13805, FCG96_17770 Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) References: UniProt: A0A444M080, UniProt: Q02MM1*PLUS #5: Protein | | Mass: 49194.168 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Gene: csy1, PA14_33330 Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) References: UniProt: Q02ML9 |
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-RNA chain , 1 types, 1 molecules K
#6: RNA chain | Mass: 19249.404 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) References: GenBank: 313291946 |
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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 | Name: Type I-F CRISPR-Csy complex with its inhibitor AcrF8 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT |
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Molecular weight | Value: 0.35 MDa / Experimental value: NO |
Source (natural) | Organism: Pseudomonas aeruginosa (bacteria) |
Source (recombinant) | Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) |
Buffer solution | pH: 8 |
Specimen | Conc.: 0.15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Details: unspecified |
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 / Cs: 2.7 mm / C2 aperture diameter: 70 µm |
Image recording | Average exposure time: 6 sec. / Electron dose: 7.6 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 6338 |
Image scans | Movie frames/image: 30 |
-Processing
Software | Name: PHENIX / Version: 1.14_3260: / Classification: refinement | ||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.42 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 91080 / Symmetry type: POINT | ||||||||||||||||||||||||
Refine LS restraints |
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