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- EMDB-35688: Cas6f of Csy-AcrIF4-dsDNA -

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Basic information

Entry
Database: EMDB / ID: EMD-35688
TitleCas6f of Csy-AcrIF4-dsDNA
Map data
Sample
  • Complex: Csy-AcrIF4-dsDNA
    • Complex: Cas6f
    • Complex: RNA
    • Complex: DNA
KeywordsIMMUNE SYSTEM-RNA-DNA complex / ANTIBIOTIC
Biological speciesPseudomonas aeruginosa (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.96 Å
AuthorsFeng Y / Zhang LX
Funding support China, 1 items
OrganizationGrant numberCountry
National Science Foundation (NSF, China)32171274 China
CitationJournal: J Biol Chem / Year: 2022
Title: Anti-CRISPR protein AcrIF4 inhibits the type I-F CRISPR-Cas surveillance complex by blocking nuclease recruitment and DNA cleavage.
Authors: Zhengyu Gao / Laixing Zhang / Zihao Ge / Hao Wang / Yourun Yue / Zhuobing Jiang / Xin Wang / Chenying Xu / Yi Zhang / Maojun Yang / Yue Feng /
Abstract: The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system provides prokaryotes with protection against mobile genetic elements such as phages. In turn, phages deploy anti- ...The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system provides prokaryotes with protection against mobile genetic elements such as phages. In turn, phages deploy anti-CRISPR (Acr) proteins to evade this immunity. AcrIF4, an Acr targeting the type I-F CRISPR-Cas system, has been reported to bind the crRNA-guided surveillance (Csy) complex. However, it remains controversial whether AcrIF4 inhibits target DNA binding to the Csy complex. Here, we present structural and mechanistic studies into AcrIF4, exploring its unique anti-CRISPR mechanism. While the Csy-AcrIF4 complex displays decreased affinity for target DNA, it is still able to bind the DNA. Our structural and functional analyses of the Csy-AcrIF4-dsDNA complex revealed that AcrIF4 binding prevents rotation of the helical bundle of the Cas8f subunit induced by dsDNA binding, therefore resulting in failure of nuclease Cas2/3 recruitment and DNA cleavage. Overall, our study provides an interesting example of attack on the nuclease recruitment event by an Acr, but not conventional mechanisms of blocking binding of target DNA.
History
DepositionMar 20, 2023-
Header (metadata) releaseJan 31, 2024-
Map releaseJan 31, 2024-
UpdateJan 31, 2024-
Current statusJan 31, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_35688.png

Downloads & links

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Map

FileDownload / File: emd_35688.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.97 Å/pix.
x 200 pix.
= 194. Å		0.97 Å/pix.
x 200 pix.
= 194. Å		0.97 Å/pix.
x 200 pix.
= 194. Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.97 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.007
Minimum - Maximum-0.008714917 - 0.027375555
Average (Standard dev.)0.00021637074 (±0.0014973796)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 194.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_35688_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_35688_half_map_2.map
Projections & Slices
AxesZYX

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Histogram (log scale)

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Sample components

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Entire : Csy-AcrIF4-dsDNA

EntireName: Csy-AcrIF4-dsDNA
Components
  • Complex: Csy-AcrIF4-dsDNA
    • Complex: Cas6f
    • Complex: RNA
    • Complex: DNA

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Supramolecule #1: Csy-AcrIF4-dsDNA

SupramoleculeName: Csy-AcrIF4-dsDNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#8
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)

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Supramolecule #2: Cas6f

SupramoleculeName: Cas6f / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#5
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)

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Supramolecule #3: RNA

SupramoleculeName: RNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #6
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)

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Supramolecule #4: DNA

SupramoleculeName: DNA / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #7-#8

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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

BufferpH: 7
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: DARK FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.7 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.96 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 93188
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER
FSC plot (resolution estimation)

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