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- PDB-7jzw: Cryo-EM structure of CRISPR-Cas surveillance complex with AcrIF4 -

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Basic information

Entry
Database: PDB / ID: 7jzw
TitleCryo-EM structure of CRISPR-Cas surveillance complex with AcrIF4
Components
  • (CRISPR type I-F/YPEST-associated protein ...) x 3
  • CRISPR repeat sequence
  • CRISPR-associated endonuclease Cas6/Csy4
  • Type I-F anti-CRISPR protein
KeywordsHYDROLASE / CRISPR
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / defense response to virus / endonuclease activity / Hydrolases; Acting on ester bonds / RNA binding
Similarity search - Function
CRISPR-associated protein Csy1 / CRISPR-associated protein (Cas_Csy1) / CRISPR-associated endoribonuclease Cas6/Csy4, subtype I-F/YPEST / CRISPR-associated endoribonuclease Cas6/Csy4, subtype I-F/YPEST superfamily / CRISPR-associated protein (Cas_Csy4) / CRISPR-associated protein Csy2 / CRISPR-associated protein (Cas_Csy2) / CRISPR-associated protein Csy3 / CRISPR-associated protein (Cas_Csy3)
Similarity search - Domain/homology
: / RNA / RNA (> 10) / Type I-F anti-CRISPR protein / CRISPR type I-F/YPEST-associated protein Csy3 / : / CRISPR type I-F/YPEST-associated protein Csy2 / Uncharacterized protein / CRISPR-associated protein Csy1 / CRISPR-associated protein Csy2 ...: / RNA / RNA (> 10) / Type I-F anti-CRISPR protein / CRISPR type I-F/YPEST-associated protein Csy3 / : / CRISPR type I-F/YPEST-associated protein Csy2 / Uncharacterized protein / CRISPR-associated protein Csy1 / CRISPR-associated protein Csy2 / CRISPR-associated protein Csy3 / CRISPR-associated endonuclease Cas6/Csy4
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria)
Pseudomonas phage sp. (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsChang, L. / Li, Z. / Gabel, C.
CitationJournal: Nucleic Acids Res / Year: 2021
Title: Structural basis for inhibition of the type I-F CRISPR-Cas surveillance complex by AcrIF4, AcrIF7 and AcrIF14.
Authors: Clinton Gabel / Zhuang Li / Heng Zhang / Leifu Chang /
Abstract: CRISPR-Cas systems are adaptive immune systems in bacteria and archaea to defend against mobile genetic elements (MGEs) and have been repurposed as genome editing tools. Anti-CRISPR (Acr) proteins ...CRISPR-Cas systems are adaptive immune systems in bacteria and archaea to defend against mobile genetic elements (MGEs) and have been repurposed as genome editing tools. Anti-CRISPR (Acr) proteins are produced by MGEs to counteract CRISPR-Cas systems and can be used to regulate genome editing by CRISPR techniques. Here, we report the cryo-EM structures of three type I-F Acr proteins, AcrIF4, AcrIF7 and AcrIF14, bound to the type I-F CRISPR-Cas surveillance complex (the Csy complex) from Pseudomonas aeruginosa. AcrIF4 binds to an unprecedented site on the C-terminal helical bundle of Cas8f subunit, precluding conformational changes required for activation of the Csy complex. AcrIF7 mimics the PAM duplex of target DNA and is bound to the N-terminal DNA vise of Cas8f. Two copies of AcrIF14 bind to the thumb domains of Cas7.4f and Cas7.6f, preventing hybridization between target DNA and the crRNA. Our results reveal structural detail of three AcrIF proteins, each binding to a different site on the Csy complex for inhibiting degradation of MGEs.
History
DepositionSep 2, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 30, 2020Provider: repository / Type: Initial release
Revision 1.1Jun 2, 2021Group: Structure summary / Category: struct / Item: _struct.title
Revision 1.2Jan 12, 2022Group: Database references / Category: citation / citation_author / database_2
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: CRISPR type I-F/YPEST-associated protein Csy1
C: CRISPR-associated endonuclease Cas6/Csy4
B: CRISPR type I-F/YPEST-associated protein Csy2
E: CRISPR type I-F/YPEST-associated protein Csy3
D: CRISPR type I-F/YPEST-associated protein Csy3
F: CRISPR type I-F/YPEST-associated protein Csy3
G: CRISPR type I-F/YPEST-associated protein Csy3
H: CRISPR type I-F/YPEST-associated protein Csy3
I: CRISPR type I-F/YPEST-associated protein Csy3
J: Type I-F anti-CRISPR protein
M: CRISPR repeat sequence


Theoretical massNumber of molelcules
Total (without water)364,32511
Polymers364,32511
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area61040 Å2
ΔGint-174 kcal/mol
Surface area112010 Å2

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Components

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CRISPR type I-F/YPEST-associated protein ... , 3 types, 8 molecules ABEDFGHI

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


Mass: 49136.133 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Gene: csy1, F7O93_18140, NCTC13619_03980 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A643HYU6, UniProt: Q02ML9*PLUS
#3: Protein CRISPR type I-F/YPEST-associated protein Csy2 / Type I-F CRISPR-associated protein Csy2


Mass: 36446.352 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, F7O93_18145, NCTC13437_01526, NCTC13619_03981, PACL_0128
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: B3G161, UniProt: Q02MM0*PLUS
#4: Protein
CRISPR type I-F/YPEST-associated protein Csy3 / CRISPR-associated protein Csy3


Mass: 37781.547 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria)
Gene: EQH76_13805, F7O93_18150, NCTC13437_01527, NCTC13619_03982
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A444M080, UniProt: Q02MM1*PLUS

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Protein , 2 types, 2 molecules CJ

#2: Protein CRISPR-associated endonuclease Cas6/Csy4 / Csy4 (Cas6f)


Mass: 21427.504 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Gene: cas6f, csy4, PA14_33300 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q02MM2, Hydrolases; Acting on ester bonds
#5: Protein Type I-F anti-CRISPR protein


Mass: 11087.513 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas phage sp. (virus) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A076FR21, UniProt: L7P7U3*PLUS

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RNA chain , 1 types, 1 molecules M

#6: RNA chain CRISPR repeat sequence


Mass: 19538.586 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: GenBank: 313291946

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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

ComponentName: CRISPR-Cas complexCRISPR / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
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: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 54 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 766782 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0124880
ELECTRON MICROSCOPYf_angle_d0.97734076
ELECTRON MICROSCOPYf_dihedral_angle_d21.4089247
ELECTRON MICROSCOPYf_chiral_restr0.0553839
ELECTRON MICROSCOPYf_plane_restr0.0074272

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