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- PDB-6w1x: Cryo-EM structure of anti-CRISPR AcrIF9, bound to the type I-F cr... -

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Basic information

Entry
Database: PDB / ID: 6w1x
TitleCryo-EM structure of anti-CRISPR AcrIF9, bound to the type I-F crRNA-guided CRISPR surveillance complex
Components
  • (CRISPR-associated protein ...) x 2
  • CRISPR-associated endonuclease Cas6/Csy4
  • RNA (60-MER)
  • Type I-F CRISPR-associated protein Csy2
  • anti-CRISPR AcrIF9
KeywordsIMMUNE SYSTEM/RNA / type I-F CRISPR RNA-guided surveillance complex / Csy complex / IMMUNE SYSTEM-RNA complex
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / endonuclease activity / defense response to virus / 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) / CRISPR type I-F/YPEST-associated protein Csy3 / Uncharacterized protein / SH3b domain-containing 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)
Proteus penneri (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsHirschi, M. / Santiago-Frangos, A. / Wilkinson, R. / Golden, S.M. / Wiedenheft, B. / Lander, G.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/Office of the DirectorDP2EB020402 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM134867 United States
National Institutes of Health/Office of the DirectorS10OD021634 United States
CitationJournal: Nat Commun / Year: 2020
Title: AcrIF9 tethers non-sequence specific dsDNA to the CRISPR RNA-guided surveillance complex.
Authors: Marscha Hirschi / Wang-Ting Lu / Andrew Santiago-Frangos / Royce Wilkinson / Sarah M Golden / Alan R Davidson / Gabriel C Lander / Blake Wiedenheft /
Abstract: Bacteria have evolved sophisticated adaptive immune systems, called CRISPR-Cas, that provide sequence-specific protection against phage infection. In turn, phages have evolved a broad spectrum of ...Bacteria have evolved sophisticated adaptive immune systems, called CRISPR-Cas, that provide sequence-specific protection against phage infection. In turn, phages have evolved a broad spectrum of anti-CRISPRs that suppress these immune systems. Here we report structures of anti-CRISPR protein IF9 (AcrIF9) in complex with the type I-F CRISPR RNA-guided surveillance complex (Csy). In addition to sterically blocking the hybridization of complementary dsDNA to the CRISPR RNA, our results show that AcrIF9 binding also promotes non-sequence-specific engagement with dsDNA, potentially sequestering the complex from target DNA. These findings highlight the versatility of anti-CRISPR mechanisms utilized by phages to suppress CRISPR-mediated immune systems.
History
DepositionMar 4, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 13, 2020Provider: repository / Type: Initial release
Revision 1.1Jun 17, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _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.year / _citation_author.identifier_ORCID
Revision 1.2Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: CRISPR-associated protein Csy1
B: Type I-F CRISPR-associated protein Csy2
C: CRISPR-associated protein Csy3
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
M: RNA (60-MER)
L: CRISPR-associated endonuclease Cas6/Csy4
I: anti-CRISPR AcrIF9
J: anti-CRISPR AcrIF9


Theoretical massNumber of molelcules
Total (without water)380,53012
Polymers380,53012
Non-polymers00
Water00
1


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

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Components

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CRISPR-associated protein ... , 2 types, 7 molecules ACDEFGH

#1: Protein CRISPR-associated protein Csy1 / Csy1 (Cas8f)


Mass: 49194.168 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Gene: csy1, PA14_33330 / Production host: Escherichia coli (E. coli) / References: UniProt: Q02ML9
#3: Protein
CRISPR-associated protein Csy3 / Csy3 (Cas7f)


Mass: 39778.594 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Gene: EQH76_13805, FCG96_17770 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A444M080, UniProt: Q02MM1*PLUS

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Protein , 3 types, 4 molecules BLIJ

#2: Protein Type I-F CRISPR-associated protein Csy2 / Csy2 (Cas5f)


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, FCG96_17775, PACL_0128
Production host: Escherichia coli (E. coli) / References: UniProt: B3G161, UniProt: Q02MM0*PLUS
#5: 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 (E. coli)
References: UniProt: Q02MM2, Hydrolases; Acting on ester bonds
#6: Protein anti-CRISPR AcrIF9


Mass: 7863.849 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Proteus penneri (bacteria) / Production host: Escherichia coli (E. coli) / References: UniProt: C0AVY5

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RNA chain , 1 types, 1 molecules M

#4: 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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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
1Anti-CRISPR AcrIF9 bound to the type I-F crRNA-guided CRISPR surveillance complexCOMPLEXall0MULTIPLE SOURCES
2Type I-F crRNA-guided CRISPR surveillance complexCOMPLEX#1-#51RECOMBINANT
3Anti-CRISPR AcrIF9COMPLEX#61RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Pseudomonas aeruginosa (bacteria)287
23Proteus penneri (bacteria)102862
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (E. coli)562
23Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
Buffer component
IDConc.FormulaBuffer-ID
120 mMHEPES1
2100 mMNaCl1
32.5 %glycerol1
41 mMTCEP1
SpecimenConc.: 2.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 36000 X / Nominal defocus max: 1200 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 13.2 sec. / Electron dose: 66 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 1653
Image scansMovie frames/image: 66

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Processing

EM softwareName: Leginon / Category: image acquisition
CTF correctionType: NONE
Particle selectionNum. of particles selected: 1571636
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 285707 / Symmetry type: POINT

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