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- EMDB-6731: Anti-CRISPR proteins AcrF1/2 bound to Csy surveillance complex wi... -

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Basic information

Entry
Database: EMDB / ID: EMD-6731
TitleAnti-CRISPR proteins AcrF1/2 bound to Csy surveillance complex with a 20nt spacer crRNA backbone region
Map dataanti-CRISPR proteins AcrF1/2 bound to Csy surveillance complex with 20nt spacer crRNA backbone region
Sample
  • Complex: anti-CRISPR proteins AcrF1/2 bound to Csy surveillance complex with a 20nt spacer sequence backbone region
    • Complex: man CRISPR-associated protein Csy3
      • Protein or peptide: CRISPR-associated protein Csy3
    • Complex: RNA
      • RNA: crRNA with 20nt spacer sequence
    • Complex: AcrF1
      • Protein or peptide: Uncharacterized protein AcrF1
Keywordsanti-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 / Uncharacterized protein / CRISPR-associated protein Csy3
Function and homology information
Biological speciesPseudomonas aeruginosa (strain UCBPP-PA14) (bacteria) / Pseudomonas phage JBD30 (virus) / Pseudomonas aeruginosa (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsPeng R / Shi Y
Funding support China, 1 items
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, 2017-
Header (metadata) releaseJan 10, 2018-
Map releaseJan 10, 2018-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5xlp
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5xlp
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6731.png

Downloads & links

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Map

FileDownload / File: emd_6731.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationanti-CRISPR proteins AcrF1/2 bound to Csy surveillance complex with 20nt spacer crRNA backbone region
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.31 Å/pix.
x 200 pix.
= 262. Å		1.31 Å/pix.
x 200 pix.
= 262. Å		1.31 Å/pix.
x 200 pix.
= 262. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.31 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.036 / Movie #1: 0.05
Minimum - Maximum-0.21832982 - 0.45560113
Average (Standard dev.)0.0007661512 (±0.009792682)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 262.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.311.311.31
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z262.000262.000262.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.2180.4560.001

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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 20nt spacer sequence backbone region
Components
  • Complex: anti-CRISPR proteins AcrF1/2 bound to Csy surveillance complex with a 20nt spacer sequence backbone region
    • Complex: man CRISPR-associated protein Csy3
      • Protein or peptide: CRISPR-associated protein Csy3
    • Complex: RNA
      • RNA: crRNA with 20nt spacer sequence
    • Complex: AcrF1
      • Protein or peptide: Uncharacterized protein AcrF1

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Supramolecule #1: anti-CRISPR proteins AcrF1/2 bound to Csy surveillance complex wi...

SupramoleculeName: anti-CRISPR proteins AcrF1/2 bound to Csy surveillance complex with a 20nt spacer sequence backbone region
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Pseudomonas aeruginosa (strain UCBPP-PA14) (bacteria)
Strain: UCBPP-PA14
Molecular weightTheoretical: 300 KDa

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Supramolecule #2: man CRISPR-associated protein Csy3

SupramoleculeName: man CRISPR-associated protein Csy3 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1

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Supramolecule #3: RNA

SupramoleculeName: RNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2

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Supramolecule #4: AcrF1

SupramoleculeName: AcrF1 / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3

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Macromolecule #1: CRISPR-associated protein Csy3

MacromoleculeName: CRISPR-associated protein Csy3 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (strain UCBPP-PA14) (bacteria)
Strain: UCBPP-PA14
Molecular weightTheoretical: 37.579273 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MSKPILSTAS VLAFERKLDP SDALMSAGAW AQRDASQEWP AVTVREKSVR GTISNRLKTK DRDPAKLDAS IQSPNLQTVD VANLPSDAD TLKVRFTLRV LGGAGTPSAC NDAAYRDKLL QTVATYVNDQ GFAELARRYA HNLANARFLW RNRVGAEAVE V RINHIRQG ...String:
MSKPILSTAS VLAFERKLDP SDALMSAGAW AQRDASQEWP AVTVREKSVR GTISNRLKTK DRDPAKLDAS IQSPNLQTVD VANLPSDAD TLKVRFTLRV LGGAGTPSAC NDAAYRDKLL QTVATYVNDQ GFAELARRYA HNLANARFLW RNRVGAEAVE V RINHIRQG EVARAWRFDA LAIGLRDFKA DAELDALAEL IASGLSGSGH VLLEVVAFAR IGDGQEVFPS QELILDKGDK KG QKSKTLY SVRDAAAIHS QKIGNALRTI DTWYPDEDGL GPIAVEPYGS VTSQGKAYRQ PKQKLDFYTL LDNWVLRDEA PAV EQQHYV IANLIRGGVF GEAEEK

UniProtKB: CRISPR-associated protein Csy3

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Macromolecule #3: Uncharacterized protein AcrF1

MacromoleculeName: Uncharacterized protein AcrF1 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas phage JBD30 (virus)
Molecular weightTheoretical: 8.824931 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString:
MKFIKYLSTA HLNYMNIAVY ENGSKIKARV ENVVNGKSVG ARDFDSTEQL ESWFYGLPGS GLGRIENAMN EISRRENP

UniProtKB: Uncharacterized protein

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Macromolecule #2: crRNA with 20nt spacer sequence

MacromoleculeName: crRNA with 20nt spacer sequence / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 15.456173 KDa
SequenceString:
CUAAGAAAUU CACGGCGGGC UUGAUGUCGU UCACUGCCGU GUAGGCAG

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.7 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average exposure time: 0.35 sec. / Average electron dose: 1.55 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0) / Number images used: 154095
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: RELION (ver. 2.0)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-5xlp:
Anti-CRISPR proteins AcrF1/2 bound to Csy surveillance complex with a 20nt spacer crRNA backbone region

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