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- PDB-5xlp: Anti-CRISPR proteins AcrF1/2 bound to Csy surveillance complex wi... -

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Basic information

Entry
Database: PDB / ID: 5xlp
TitleAnti-CRISPR proteins AcrF1/2 bound to Csy surveillance complex with a 20nt spacer crRNA backbone region
Components
  • CRISPR-associated protein Csy3
  • Uncharacterized protein AcrF1
  • crRNA with 20nt spacer sequence
KeywordsIMMUNE SYSTEM/RNA / anti-CRISPR proteins / Csy complex / Type I-F CRISPR/Cas system / IMMUNE SYSTEM-RNA complex
Function / homology: / Anti-CRISPR protein Acr30-35/AcrF1 / CRISPR-associated protein Csy3 / CRISPR-associated protein (Cas_Csy3) / defense response to virus / RNA / RNA (> 10) / Uncharacterized protein / CRISPR-associated protein Csy3
Function and homology information
Biological speciesPseudomonas aeruginosa (bacteria)
Pseudomonas phage JBD30 (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsPeng, R. / Shi, Y. / Gao, G.F.
Funding support China, 1items
OrganizationGrant numberCountry
China Ministry of Science and Technology National 973 Project2013CB531502 China
CitationJournal: Cell Res / Year: 2017
Title: Alternate binding modes of anti-CRISPR viral suppressors AcrF1/2 to Csy surveillance complex revealed by cryo-EM structures.
Authors: Ruchao Peng / Ying Xu / Tengfei Zhu / Ningning Li / Jianxun Qi / Yan Chai / Min Wu / Xinzheng Zhang / Yi Shi / Peiyi Wang / Jiawei Wang / Ning Gao / George Fu Gao /
Abstract: Bacteriophages encode anti-CRISPR suppressors to counteract the CRISPR/Cas immunity of their bacterial hosts, thus facilitating their survival and replication. Previous studies have shown that two ...Bacteriophages encode anti-CRISPR suppressors to counteract the CRISPR/Cas immunity of their bacterial hosts, thus facilitating their survival and replication. Previous studies have shown that two phage-encoded anti-CRISPR proteins, AcrF1 and AcrF2, suppress the type I-F CRISPR/Cas system of Pseudomonas aeruginosa by preventing target DNA recognition by the Csy surveillance complex, but the precise underlying mechanism was unknown. Here we present the structure of AcrF1/2 bound to the Csy complex determined by cryo-EM single-particle reconstruction. By structural analysis, we found that AcrF1 inhibits target DNA recognition of the Csy complex by interfering with base pairing between the DNA target strand and crRNA spacer. In addition, multiple copies of AcrF1 bind to the Csy complex with different modes when working individually or cooperating with AcrF2, which might exclude target DNA binding through different mechanisms. Together with previous reports, we provide a comprehensive working scenario for the two anti-CRISPR suppressors, AcrF1 and AcrF2, which silence CRISPR/Cas immunity by targeting the Csy surveillance complex.
History
DepositionMay 11, 2017Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jan 10, 2018Provider: repository / Type: Initial release
Revision 1.1Mar 27, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
C: CRISPR-associated protein Csy3
D: CRISPR-associated protein Csy3
E: CRISPR-associated protein Csy3
F: CRISPR-associated protein Csy3
K: crRNA with 20nt spacer sequence
M: Uncharacterized protein AcrF1


Theoretical massNumber of molelcules
Total (without water)174,5986
Polymers174,5986
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, homology, microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area18050 Å2
ΔGint-113 kcal/mol
Surface area64960 Å2

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Components

#1: Protein
CRISPR-associated protein Csy3


Mass: 37579.273 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (strain UCBPP-PA14) (bacteria)
Strain: UCBPP-PA14 / Gene: csy3, csy1-3, PA14_33310
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Strain (production host): BL21-Gold(DE3)pLysS AG / References: UniProt: Q02MM1
#2: RNA chain crRNA with 20nt spacer sequence


Mass: 15456.173 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)
Strain (production host): BL21-Gold(DE3)pLysS AG
#3: Protein Uncharacterized protein AcrF1


Mass: 8824.931 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas phage JBD30 (virus) / Gene: JBD30_035
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Strain (production host): BL21-Gold(DE3)pLysS AG / References: UniProt: L7P7M1

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1anti-CRISPR proteins AcrF1/2 bound to Csy surveillance complex with a 20nt spacer sequence backbone regionCOMPLEXall0RECOMBINANT
2man CRISPR-associated protein Csy3COMPLEX#11RECOMBINANT
3RNACOMPLEX#21RECOMBINANT
4AcrF1COMPLEX#31RECOMBINANT
Molecular weightValue: 0.3 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
21Pseudomonas aeruginosa (strain UCBPP-PA14) (bacteria)208963UCBPP-PA14
31Pseudomonas aeruginosa (bacteria)287
41Pseudomonas phage D301223260
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)866768
31Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)866768
41Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)866768
Buffer solutionpH: 7.5
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Image recordingAverage exposure time: 0.35 sec. / Electron dose: 1.55 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansMovie frames/image: 38

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM software
IDNameVersionCategory
1RELION2particle selection
4CTF3CTF correction
7Chimera2015-04-15model fitting
9RELION2initial Euler assignment
10RELION2final Euler assignment
11RELION2classification
12RELION23D reconstruction
13PHENIX1.11model refinement
CTF correctionType: NONE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 154095 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00910142
ELECTRON MICROSCOPYf_angle_d1.35613963
ELECTRON MICROSCOPYf_dihedral_angle_d13.285999
ELECTRON MICROSCOPYf_chiral_restr0.0641691
ELECTRON MICROSCOPYf_plane_restr0.0091783

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