|Entry||Database: EMDB / ID: 6731|
|Title||Anti-CRISPR proteins AcrF1/2 bound to Csy surveillance complex with a 20nt spacer crRNA backbone region|
|Map data||anti-CRISPR proteins AcrF1/2 bound to Csy surveillance complex with 20nt spacer crRNA backbone region|
|Sample||anti-CRISPR proteins AcrF1/2 bound to Csy surveillance complex with a 20nt spacer sequence backbone region|
|Function/homology||CRISPR-associated protein Csy3 / CRISPR-associated protein (Cas_Csy3) / Uncharacterized protein / CRISPR-associated protein Csy3|
Function and homology information
|Source||Pseudomonas aeruginosa (strain ucbpp-pa14) / / bacteria|
|Method||Cryo EM / single particle reconstruction / 4.2 Å resolution|
|Authors||Peng R / Shi Y|
|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.
|Validation Report||PDB-ID: 5xlp|
SummaryFull reportAbout validation report
|Date||Deposition: May 11, 2017 / Header (metadata) release: Jan 10, 2018 / Map release: Jan 10, 2018 / Last update: Jan 10, 2018|
Downloads & links
|File||emd_6731.map.gz (map file in CCP4 format, 32001 KB)|
|Projections & slices|
Images are generated by Spider package.
|Voxel size||X=Y=Z: 1.31 Å|
CCP4 map header:
+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 sequence backbone region|
Number of components: 7
|Mass||Experimental: 300 kDa|
+Component #1: protein, anti-CRISPR proteins AcrF1/2 bound to Csy surveillance c...
|Protein||Name: anti-CRISPR proteins AcrF1/2 bound to Csy surveillance complex with a 20nt spacer sequence backbone region|
Recombinant expression: No
|Mass||Experimental: 300 kDa|
|Source||Species: Pseudomonas aeruginosa (strain UCBPP-PA14) / Strain: UCBPP-PA14|
|Source (engineered)||Expression System: Escherichia coli 'BL21-Gold(DE3)pLysS AG'|
+Component #2: protein, man CRISPR-associated protein Csy3
|Protein||Name: man CRISPR-associated protein Csy3 / Recombinant expression: No|
+Component #3: protein, RNA
|Protein||Name: RNA / Recombinant expression: No|
+Component #4: protein, AcrF1
|Protein||Name: AcrF1 / Recombinant expression: No|
+Component #5: protein, CRISPR-associated protein Csy3
|Protein||Name: CRISPR-associated protein Csy3 / Recombinant expression: No|
|Mass||Theoretical: 37.579273 kDa|
|Source (engineered)||Expression System: Pseudomonas aeruginosa (strain UCBPP-PA14)|
+Component #6: nucleic-acid, crRNA with 20nt spacer sequence
|Nucleic-acid||Name: crRNA with 20nt spacer sequence / Class: RNA / Structure: OTHER / Synthetic: No|
CUAAGAAAUU CACGGCGGGC UUGAUGUCGU UCACUGCCGU GUAGGCAG
|Mass||Theoretical: 15.456173 kDa|
+Component #7: protein, Uncharacterized protein AcrF1
|Protein||Name: Uncharacterized protein AcrF1 / Recombinant expression: No|
|Mass||Theoretical: 8.824931 kDa|
|Source (engineered)||Expression System: Pseudomonas phage JBD30|
|Specimen||Specimen state: particle / Method: Cryo EM|
|Sample solution||Specimen conc.: 0.7 mg/ml / pH: 7.5|
|Vitrification||Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 100 %|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Imaging||Microscope: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1.55 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Cs: 2.7 mm / Imaging mode: BRIGHT FIELD|
|Specimen Holder||Model: OTHER|
|Camera||Detector: GATAN K2 (4k x 4k)|
|Processing||Method: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 154095|
|3D reconstruction||Software: RELION / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF|
|FSC plot (resolution assessment)|
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