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Yorodumi- EMDB-6728: Anti-CRISPR protein AcrF1/2 bound to Csy surveillance complex wit... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-6728 | |||||||||
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Title | Anti-CRISPR protein AcrF1/2 bound to Csy surveillance complex with a 20nt spacer crRNA | |||||||||
Map data | AcrF1/2-Csy20nt full component reconstruction | |||||||||
Sample |
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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 species | Pseudomonas aeruginosa (bacteria) / Pseudomonas phage D3 (virus) / Pseudomonas phage 73 (virus) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 5.3 Å | |||||||||
Authors | Peng R / Shi Y / Gao FG | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_6728.map.gz | 2.2 MB | EMDB map data format | |
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Header (meta data) | emd-6728-v30.xml emd-6728.xml | 17.1 KB 17.1 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_6728_fsc.xml | 7.1 KB | Display | FSC data file |
Images | emd_6728.png | 159.1 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-6728 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-6728 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_6728.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | AcrF1/2-Csy20nt full component reconstruction | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.31 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : anti-CRISPR proteins AcrF1/2 bound to Csy surveillance complex wi...
Entire | Name: anti-CRISPR proteins AcrF1/2 bound to Csy surveillance complex with a 20nt spacer crRNA |
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Components |
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-Supramolecule #1: anti-CRISPR proteins AcrF1/2 bound to Csy surveillance complex wi...
Supramolecule | Name: 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 |
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Source (natural) | Organism: Pseudomonas aeruginosa (bacteria) |
Recombinant expression | Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) |
Molecular weight | Experimental: 300 KDa |
-Macromolecule #1: Csy1
Macromolecule | Name: Csy1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Pseudomonas aeruginosa (bacteria) |
Recombinant expression | Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) |
Sequence | String: 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 |
-Macromolecule #2: Csy2
Macromolecule | Name: Csy2 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Pseudomonas aeruginosa (bacteria) |
Recombinant expression | Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) |
Sequence | String: 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 |
-Macromolecule #3: Csy3
Macromolecule | Name: Csy3 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO |
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Source (natural) | Organism: Pseudomonas aeruginosa (bacteria) |
Recombinant expression | Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) |
Sequence | String: 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 |
-Macromolecule #4: Csy4
Macromolecule | Name: Csy4 / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO |
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Source (natural) | Organism: Pseudomonas aeruginosa (bacteria) |
Recombinant expression | Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) |
Sequence | String: MDHYLDIRLR PDPEFPPAQL MSVLFGKLHQ ALVAQGGDRI GVSFPDLDES RSRLGERLRI HASADDLRAL LARPWLEGLR DHLQFGEPAV VPHPTPYRQV SRVQAKSNPE RLRRRLMRRH DLSEEEARKR IPDTVARALD LPFVTLRSQS TGQHFRLFIR HGPLQVTAEE GGFTCYGLSK GGFVPWF |
-Macromolecule #6: AcrF1
Macromolecule | Name: AcrF1 / type: protein_or_peptide / ID: 6 / Enantiomer: LEVO |
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Source (natural) | Organism: Pseudomonas phage D3 (virus) |
Recombinant expression | Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) |
Sequence | String: MKFIKYLSTA HLNYMNIAVY ENGSKIKARV ENVVNGKSVG ARDFDSTEQL ESWFYGLPGS GLGRIENAMN EISRRENP |
-Macromolecule #7: AcrF2
Macromolecule | Name: AcrF2 / type: protein_or_peptide / ID: 7 / Enantiomer: LEVO |
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Source (natural) | Organism: Pseudomonas phage 73 (virus) |
Recombinant expression | Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) |
Sequence | String: MTKTAQMIAQ QHKDTVAACE AAEAIAIAKD QVWDGEGYTK YTFDDNSVLI QSGTTQYAMD ADDADSIKGY ADWLDDEARS AEASEIERLL ESVEEE |
-Macromolecule #5: crRNA with 20nt spacer
Macromolecule | Name: crRNA with 20nt spacer / type: rna / ID: 5 |
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Source (natural) | Organism: Pseudomonas aeruginosa (bacteria) |
Sequence | String: CUAAGAAAUU CACGGCGGGC UUGAUGUCGU UCACUGCCGU GUAGGCAG |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.7 mg/mL |
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Buffer | pH: 7.5 |
Grid | Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm |
Image recording | Film 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 |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
-Atomic model buiding 1
Refinement | Space: REAL / Protocol: FLEXIBLE FIT |
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