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- EMDB-6729: Anti-CRISPR proteins AcrF1/2 bound to Csy surveillance complex wi... -

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Basic information

Entry
Database: EMDB / ID: 6729
TitleAnti-CRISPR proteins AcrF1/2 bound to Csy surveillance complex with a 32nt spacer crRNA backbone region
Map data
Sampleanti-CRISPR proteins AcrF1/2 bound to Csy surveillance complex with a 32nt spacer crRNA backbone region
Function/homologyCRISPR-associated protein Csy3 / CRISPR-associated protein (Cas_Csy3) / Uncharacterized protein / CRISPR-associated protein Csy3
Function and homology information
SourcePseudomonas aeruginosa (strain ucbpp-pa14) / bacteria /
Methodsingle particle reconstruction, at 3.8 Å resolution
AuthorsPeng R / Shi Y / Gao GF
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.
Validation ReportPDB-ID: 5xlo

SummaryFull reportAbout validation report
DateDeposition: May 11, 2017 / Header (metadata) release: Jan 10, 2018 / Map release: Jan 10, 2018 / Last update: Jan 10, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.033
  • Imaged by UCSF CHIMERA
  • Download
  • Surface view colored by radius
  • Surface level: 0.033
  • Imaged by UCSF CHIMERA
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  • Surface view with fitted model
  • Atomic models: : PDB-5xlo
  • Surface level: 0.033
  • Imaged by UCSF CHIMERA
  • Download
3D viewer
Supplemental images

Downloads & links

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Map

Fileemd_6729.map.gz (map file in CCP4 format, 23329 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
180 pix
1.3 Å/pix.
= 234. Å
180 pix
1.3 Å/pix.
= 234. Å
180 pix
1.3 Å/pix.
= 234. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider package.

Voxel sizeX=Y=Z: 1.3 Å
Density
Contour Level:0.033 (by author), 0.033 (movie #1):
Minimum - Maximum-0.2249125 - 0.3496538
Average (Standard dev.)0.00044728076 (0.009759382)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions180180180
Origin000
Limit179179179
Spacing180180180
CellA=B=C: 233.99998 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.31.31.3
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z234.000234.000234.000
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS180180180
D min/max/mean-0.2250.3500.000

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Supplemental data

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Sample components

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Entire anti-CRISPR proteins AcrF1/2 bound to Csy surveillance complex wi...

EntireName: anti-CRISPR proteins AcrF1/2 bound to Csy surveillance complex with a 32nt spacer crRNA backbone region
Number of components: 7
MassExperimental: 400 kDa

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Component #1: protein, anti-CRISPR proteins AcrF1/2 bound to Csy surveillance c...

ProteinName: anti-CRISPR proteins AcrF1/2 bound to Csy surveillance complex with a 32nt spacer crRNA backbone region
Recombinant expression: No
MassExperimental: 400 kDa
SourceSpecies: Pseudomonas aeruginosa (strain ucbpp-pa14) / bacteria /
Strain: UCBPP-PA14
Source (engineered)Expression System: Escherichia coli 'bl21-gold(de3)plyss ag' / bacteria / / image: Escherichia coli

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Component #2: protein, CRISPR-associated protein Csy3

ProteinName: CRISPR-associated protein Csy3 / Recombinant expression: No

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Component #3: protein, crRNA with 20nt spacer sequence

ProteinName: crRNA with 20nt spacer sequence / Recombinant expression: No

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Component #4: protein, AcrF1

ProteinName: AcrF1 / Recombinant expression: No

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Component #5: protein, CRISPR-associated protein Csy3

ProteinName: CRISPR-associated protein Csy3 / Recombinant expression: No
MassTheoretical: 37.579273 kDa
Source (engineered)Expression System: Pseudomonas aeruginosa (strain ucbpp-pa14) / bacteria /
Strain: UCBPP-PA14

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Component #6: nucleic-acid, crRNA with 32nt spacer sequence

Nucleic-acidName: crRNA with 32nt spacer sequence / Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
CUAAGAAAUU CACGGCGGGC UUGAUGUCCG CGUCUACCUG GUUCACUGCC GUGUAGGCAG
MassTheoretical: 19.265404 kDa

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Component #7: protein, Uncharacterized protein AcrF1

ProteinName: Uncharacterized protein AcrF1 / Recombinant expression: No
MassTheoretical: 8.824931 kDa
Source (engineered)Expression System: Pseudomonas phage JBD30 / virus

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Experimental details

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Sample preparation

Specimen stateparticle
Sample solutionSpecimen conc.: 0.4 mg/ml / pH: 7.5
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 155099
3D reconstructionSoftware: RELION / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution assessment)

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Atomic model buiding

Output model

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