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- PDB-7eqg: Structure of Csy-AcrIF5 -

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Basic information

Entry
Database: PDB / ID: 7eqg
TitleStructure of Csy-AcrIF5
Components
  • (DNA (54-MER)) x 2
  • AcrIF5
  • CRISPR type I-F/YPEST-associated protein Csy2
  • CRISPR-associated protein Csy3
  • RNA (60-MER)
  • Type I-F CRISPR-associated protein Csy1
  • type I-F CRISPR-associated endoribonuclease Cas6/Csy4
KeywordsIMMUNE SYSTEM/RNA/DNA / complex / inhibitor / IMMUNE SYSTEM / 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 / : / Uncharacterized protein
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsZhang, L. / Feng, Y.
Funding support China, 1items
OrganizationGrant numberCountry
National Science Foundation (NSF, China)31822012 China
CitationJournal: Nat Chem Biol / Year: 2022
Title: AcrIF5 specifically targets DNA-bound CRISPR-Cas surveillance complex for inhibition.
Authors: Yongchao Xie / Laixing Zhang / Zhengyu Gao / Peipei Yin / Hao Wang / Hang Li / Zeliang Chen / Yi Zhang / Maojun Yang / Yue Feng /
Abstract: CRISPR-Cas systems are prokaryotic antiviral systems, and phages use anti-CRISPR proteins (Acrs) to inactivate these systems. Here we present structural and functional analyses of AcrIF5, exploring ...CRISPR-Cas systems are prokaryotic antiviral systems, and phages use anti-CRISPR proteins (Acrs) to inactivate these systems. Here we present structural and functional analyses of AcrIF5, exploring its unique anti-CRISPR mechanism. AcrIF5 shows binding specificity only for the target DNA-bound form of the crRNA-guided surveillance (Csy) complex, but not the apo Csy complex from the type I-F CRISPR-Cas system. We solved the structure of the Csy-dsDNA-AcrIF5 complex, revealing that the conformational changes of the Csy complex caused by dsDNA binding dictate the binding specificity for the Csy-dsDNA complex by AcrIF5. Mechanistically, five AcrIF5 molecules bind one Csy-dsDNA complex, which destabilizes the helical bundle domain of Cas8f, thus preventing subsequent Cas2/3 recruitment. AcrIF5 exists in symbiosis with AcrIF3, which blocks Cas2/3 recruitment. This attack on the recruitment event stands in contrast to the conventional mechanisms of blocking binding of target DNA. Overall, our study reveals an unprecedented mechanism of CRISPR-Cas inhibition by AcrIF5.
History
DepositionMay 1, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 9, 2022Provider: repository / Type: Initial release
Revision 1.1Mar 30, 2022Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Jun 8, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Jun 5, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

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Assembly

Deposited unit
C: CRISPR type I-F/YPEST-associated protein Csy2
D: CRISPR-associated protein Csy3
E: CRISPR-associated protein Csy3
F: CRISPR-associated protein Csy3
G: CRISPR-associated protein Csy3
H: CRISPR-associated protein Csy3
I: CRISPR-associated protein Csy3
J: AcrIF5
K: AcrIF5
L: type I-F CRISPR-associated endoribonuclease Cas6/Csy4
M: RNA (60-MER)
N: DNA (54-MER)
P: AcrIF5
Q: AcrIF5
R: AcrIF5
B: Type I-F CRISPR-associated protein Csy1
O: DNA (54-MER)


Theoretical massNumber of molelcules
Total (without water)427,93517
Polymers427,93517
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area79950 Å2
ΔGint-227 kcal/mol
Surface area135140 Å2

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Components

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Protein , 5 types, 14 molecules CDEFGHIJKPQRLB

#1: 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, ALP65_00953, EQH76_13810, NCTC13437_01526, PACL_0128
Production host: Escherichia coli (E. coli) / References: UniProt: B3G161
#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) / Gene: IPC1505_30680, IPC36_28835 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A659BSG0
#3: Protein
AcrIF5


Mass: 8531.865 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Gene: IPC1164_31450 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A7M3A7R4
#4: Protein type I-F CRISPR-associated endoribonuclease Cas6/Csy4


Mass: 21429.477 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)
#7: Protein Type I-F CRISPR-associated protein Csy1


Mass: 49313.254 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Gene: csy1, ALP65_00954, IPC1505_30690 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A3A8DDU9

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RNA chain , 1 types, 1 molecules M

#5: RNA chain RNA (60-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

#6: DNA chain DNA (54-MER)


Mass: 16708.691 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Pseudomonas aeruginosa (bacteria)
#8: DNA chain DNA (54-MER)


Mass: 16574.561 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Pseudomonas aeruginosa (bacteria)

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Details

Sequence detailsThe reference sequence database of chain L is WP_004343806.1 in GenBank.

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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-AcrIF5COMPLEXall0RECOMBINANT
2Csy-AcrIF5COMPLEX#1-#5, #71RECOMBINANT
3DNACOMPLEX#6, #81SYNTHETIC
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
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: FLOOD BEAM
Electron lensMode: DARK FIELD
Image recordingElectron dose: 20 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: NONE
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 173096 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00526835
ELECTRON MICROSCOPYf_angle_d0.81736967
ELECTRON MICROSCOPYf_dihedral_angle_d21.14727
ELECTRON MICROSCOPYf_chiral_restr0.054203
ELECTRON MICROSCOPYf_plane_restr0.0064439

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