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- EMDB-6728: Anti-CRISPR protein AcrF1/2 bound to Csy surveillance complex wit... -

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Basic information

Entry
Database: EMDB / ID: EMD-6728
TitleAnti-CRISPR protein AcrF1/2 bound to Csy surveillance complex with a 20nt spacer crRNA
Map dataAcrF1/2-Csy20nt full component reconstruction
Sample
  • Complex: anti-CRISPR proteins AcrF1/2 bound to Csy surveillance complex with a 20nt spacer crRNA
    • Protein or peptide: Csy1
    • Protein or peptide: Csy2
    • Protein or peptide: Csy3
    • Protein or peptide: Csy4
    • RNA: crRNA with 20nt spacer
    • Protein or peptide: AcrF1
    • Protein or peptide: AcrF2
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 (bacteria) / Pseudomonas phage D3 (virus) / Pseudomonas phage 73 (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.3 Å
AuthorsPeng R / Shi Y / Gao FG
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-
UpdateJan 10, 2018-
Current statusJan 10, 2018Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.045
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.045
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6728.png

Downloads & links

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Map

FileDownload / File: emd_6728.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAcrF1/2-Csy20nt full component reconstruction
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: 0.045 / Movie #1: 0.045
Minimum - Maximum-0.12020987 - 0.17177638
Average (Standard dev.)0.0010149188 (±0.008057097)
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-0.1200.1720.001

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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 crRNA
Components
  • Complex: anti-CRISPR proteins AcrF1/2 bound to Csy surveillance complex with a 20nt spacer crRNA
    • Protein or peptide: Csy1
    • Protein or peptide: Csy2
    • Protein or peptide: Csy3
    • Protein or peptide: Csy4
    • RNA: crRNA with 20nt spacer
    • Protein or peptide: AcrF1
    • Protein or peptide: AcrF2

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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 crRNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#7
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Molecular weightExperimental: 300 KDa

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Macromolecule #1: Csy1

MacromoleculeName: Csy1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MTSPLPTPTW QELRQFIESF IQERLQGKLD KLQPDEDDKR QTLLATHRRE AWLADAARRV GQLQLVTHTL KPIHPDARGS NLHSLPQAPG QPGLAGSHEL GDRLVSDVVG NAAALDVFKF LSLQYQGKNL LNWLTEDSAE ALQALSDNAE QAREWRQAFI GITTVKGAPA ...String:
MTSPLPTPTW QELRQFIESF IQERLQGKLD KLQPDEDDKR QTLLATHRRE AWLADAARRV GQLQLVTHTL KPIHPDARGS NLHSLPQAPG QPGLAGSHEL GDRLVSDVVG NAAALDVFKF LSLQYQGKNL LNWLTEDSAE ALQALSDNAE QAREWRQAFI GITTVKGAPA SHSLAKQLYF PLPGSGYHLL APLFPTSLVH HVHALLREAR FGDAAKAARE ARSRQESWPH GFSEYPNLAI QKFGGTKPQN ISQLNNERRG ENWLLPSLPP NWQRQNVNAP MRHSSVFEHD FGRTPEVSRL TRTLQRFLAK TVHNNLAIRQ RRAQLVAQIC DEALQYAARL RELEPGWSAT PGCQLHDAEQ LWLDPLRAQT DETFLQRRLR GDWPAEVGNR FANWLNRAVS SDSQILGSPE AAQWSQELSK ELTMFKEILE DERD

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Macromolecule #2: Csy2

MacromoleculeName: Csy2 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MSVTDPEALL LLPRLSIQNA NAISSPLTWG FPSPGAFTGF VHALQRRVGI SLDIELDGVG IVCHRFEAQI SQPAGKRTKV FNLTRNPLNR DGSTAAIVEE GRAHLEVSLL LGVHGDGLDD HPAQEIARQV QEQAGAMRLA GGSILPWCNE RFPAPNAELL MLGGSDEQRR ...String:
MSVTDPEALL LLPRLSIQNA NAISSPLTWG FPSPGAFTGF VHALQRRVGI SLDIELDGVG IVCHRFEAQI SQPAGKRTKV FNLTRNPLNR DGSTAAIVEE GRAHLEVSLL LGVHGDGLDD HPAQEIARQV QEQAGAMRLA GGSILPWCNE RFPAPNAELL MLGGSDEQRR KNQRRLTRRL LPGFALVSRE ALLQQHLETL RTTLPEATTL DALLDLCRIN FEPPATSSEE EASPPDAAWQ VRDKPGWLVP IPAGYNALSP LYLPGEVRNA RDRETPLRFV ENLFGLGEWL SPHRVAALSD LLWYHHAEPD KGLYRWSTPR FVEHAIA

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Macromolecule #3: Csy3

MacromoleculeName: Csy3 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MSKPILSTAS VLAFERKLDP SDALMSAGAW AQRDASQEWP AVTVREKSVR GTISNRLKTK DRDPAKLDAS IQSPNLQTVD VANLPSDADT LKVRFTLRVL GGAGTPSACN DAAYRDKLLQ TVATYVNDQG FAELARRYAH NLANARFLWR NRVGAEAVEV RINHIRQGEV ...String:
MSKPILSTAS VLAFERKLDP SDALMSAGAW AQRDASQEWP AVTVREKSVR GTISNRLKTK DRDPAKLDAS IQSPNLQTVD VANLPSDADT LKVRFTLRVL GGAGTPSACN DAAYRDKLLQ TVATYVNDQG FAELARRYAH NLANARFLWR NRVGAEAVEV RINHIRQGEV ARAWRFDALA IGLRDFKADA ELDALAELIA SGLSGSGHVL LEVVAFARIG DGQEVFPSQE LILDKGDKKG QKSKTLYSVR DAAAIHSQKI GNALRTIDTW YPDEDGLGPI AVEPYGSVTS QGKAYRQPKQ KLDFYTLLDN WVLRDEAPAV EQQHYVIANL IRGGVFGEAE EK

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Macromolecule #4: Csy4

MacromoleculeName: Csy4 / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString:
MDHYLDIRLR PDPEFPPAQL MSVLFGKLHQ ALVAQGGDRI GVSFPDLDES RSRLGERLRI HASADDLRAL LARPWLEGLR DHLQFGEPAV VPHPTPYRQV SRVQAKSNPE RLRRRLMRRH DLSEEEARKR IPDTVARALD LPFVTLRSQS TGQHFRLFIR HGPLQVTAEE GGFTCYGLSK GGFVPWF

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Macromolecule #6: AcrF1

MacromoleculeName: AcrF1 / type: protein_or_peptide / ID: 6 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas phage D3 (virus)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString:
MKFIKYLSTA HLNYMNIAVY ENGSKIKARV ENVVNGKSVG ARDFDSTEQL ESWFYGLPGS GLGRIENAMN EISRRENP

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Macromolecule #7: AcrF2

MacromoleculeName: AcrF2 / type: protein_or_peptide / ID: 7 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas phage 73 (virus)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString:
MTKTAQMIAQ QHKDTVAACE AAEAIAIAKD QVWDGEGYTK YTFDDNSVLI QSGTTQYAMD ADDADSIKGY ADWLDDEARS AEASEIERLL ESVEEE

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Macromolecule #5: crRNA with 20nt spacer

MacromoleculeName: crRNA with 20nt spacer / type: rna / ID: 5
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
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 - 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

CTF correctionSoftware - Name: CTFFIND (ver. 3.0)
Startup modelType of model: INSILICO MODEL / In silico model: gauss sphere
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 5.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0) / Number images used: 53974
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

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