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- PDB-7yhs: Structure of Csy-AcrIF4-dsDNA -

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Basic information

Entry
Database: PDB / ID: 7yhs
TitleStructure of Csy-AcrIF4-dsDNA
Components
  • AcrIF4
  • CRISPR type I-F/YPEST-associated protein Csy2
  • CRISPR-associated protein Csy3
  • Csy4
  • DNA (39-MER)
  • DNA (5'-D(P*AP*GP*CP*AP*GP*CP*TP*GP*CP*AP*CP*C)-3')
  • RNA (58-MER)
  • Type I-F CRISPR-associated protein Csy1
KeywordsIMMUNE SYSTEM/RNA/DNA / IMMUNE SYSTEM-RNA-DNA complex
Function / homology
Function and homology information


CRISPR-associated protein Csy1 / CRISPR-associated protein (Cas_Csy1) / CRISPR-associated protein Csy2 / CRISPR-associated protein (Cas_Csy2) / CRISPR-associated protein Csy3 / CRISPR-associated protein (Cas_Csy3)
Similarity search - Domain/homology
: / : / DNA / DNA (> 10) / RNA / RNA (> 10) / Uncharacterized protein / CRISPR-associated protein Csy3 / CRISPR type I-F/YPEST-associated protein Csy2
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.37 Å
AuthorsFeng, Y. / Zhang, L.X.
Funding support China, 1items
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
DepositionJul 14, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 29, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Type I-F CRISPR-associated protein Csy1
C: CRISPR-associated protein Csy3
D: CRISPR-associated protein Csy3
E: CRISPR type I-F/YPEST-associated protein Csy2
F: CRISPR-associated protein Csy3
G: CRISPR-associated protein Csy3
H: CRISPR-associated protein Csy3
I: CRISPR-associated protein Csy3
J: AcrIF4
L: Csy4
M: RNA (58-MER)
N: DNA (39-MER)
O: DNA (5'-D(P*AP*GP*CP*AP*GP*CP*TP*GP*CP*AP*CP*C)-3')


Theoretical massNumber of molelcules
Total (without water)396,39113
Polymers396,39113
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 5 types, 10 molecules ACDFGHIEJL

#1: Protein Type I-F CRISPR-associated protein Csy1


Mass: 49313.254 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: WP_004343803.1 / Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Gene: csy1 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A3A8DDU9
#2: Protein
CRISPR-associated protein Csy3


Mass: 37623.324 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Production host: Escherichia coli (E. coli) / References: UniProt: A0A659BSG0
#3: Protein CRISPR type I-F/YPEST-associated protein Csy2 / Type I-F CRISPR-associated protein Csy2


Mass: 36244.074 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Gene: csy2 / Production host: Escherichia coli (E. coli) / References: UniProt: B3G161
#4: Protein AcrIF4


Mass: 11115.526 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: WP_016068584.1 / Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Production host: Escherichia coli (E. coli)
#5: Protein Csy4


Mass: 21429.477 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: WP_004343806.1 / Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Production host: Escherichia coli (E. coli)

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RNA chain , 1 types, 1 molecules M

#6: RNA chain RNA (58-MER)


Mass: 19265.404 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Production host: Escherichia coli (E. coli) / References: GenBank: 313291946

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DNA chain , 2 types, 2 molecules NO

#7: DNA chain DNA (39-MER)


Mass: 16708.691 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Production host: Escherichia coli (E. coli) / References: GenBank: 1910818228
#8: DNA chain DNA (5'-D(P*AP*GP*CP*AP*GP*CP*TP*GP*CP*AP*CP*C)-3')


Mass: 16574.561 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Production host: Escherichia coli (E. coli) / References: GenBank: 1910818228

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Csy-AcrIF4-dsDNACOMPLEXall0RECOMBINANT
2Csy-AcrIF4COMPLEX#1-#51RECOMBINANT
3RNACOMPLEX#61RECOMBINANT
4DNACOMPLEX#7-#81RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Pseudomonas aeruginosa (bacteria)287
32Pseudomonas aeruginosa (bacteria)287
43Pseudomonas aeruginosa (bacteria)287
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli (E. coli)562
32Escherichia coli (E. coli)562
43Escherichia coli (E. coli)562
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: DARK FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1700 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.37 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 117510 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00525295
ELECTRON MICROSCOPYf_angle_d0.85334865
ELECTRON MICROSCOPYf_dihedral_angle_d17.6514419
ELECTRON MICROSCOPYf_chiral_restr0.0493975
ELECTRON MICROSCOPYf_plane_restr0.0074195

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