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- EMDB-33837: Structure of Csy-AcrIF4-dsDNA -

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Basic information

Entry
Database: EMDB / ID: EMD-33837
TitleStructure of Csy-AcrIF4-dsDNA
Map data
Sample
  • Complex: Csy-AcrIF4-dsDNA
    • Complex: Csy-AcrIF4
      • Protein or peptide: Type I-F CRISPR-associated protein Csy1
      • Protein or peptide: CRISPR-associated protein Csy3
      • Protein or peptide: CRISPR type I-F/YPEST-associated protein Csy2
      • Protein or peptide: AcrIF4
      • Protein or peptide: Csy4
    • Complex: RNA
      • RNA: RNA (58-MER)
    • Complex: DNA
      • DNA: DNA (39-MER)
      • DNA: DNA (5'-D(P*AP*GP*CP*AP*GP*CP*TP*GP*CP*AP*CP*C)-3')
Function / homologyCRISPR-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) / Uncharacterized protein / CRISPR-associated protein Csy3 / CRISPR type I-F/YPEST-associated protein Csy2
Function and homology information
Biological speciesPseudomonas aeruginosa (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.37 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
DepositionJul 14, 2022-
Header (metadata) releaseMar 29, 2023-
Map releaseMar 29, 2023-
UpdateMar 29, 2023-
Current statusMar 29, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33837.png

Downloads & links

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Map

FileDownload / File: emd_33837.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.97 Å
Density
Contour LevelBy AUTHOR: 0.06
Minimum - Maximum-0.05292088 - 2.3923914
Average (Standard dev.)0.00398018 (±0.04177604)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 232.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_33837_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_33837_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_33837_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

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: Csy-AcrIF4
      • Protein or peptide: Type I-F CRISPR-associated protein Csy1
      • Protein or peptide: CRISPR-associated protein Csy3
      • Protein or peptide: CRISPR type I-F/YPEST-associated protein Csy2
      • Protein or peptide: AcrIF4
      • Protein or peptide: Csy4
    • Complex: RNA
      • RNA: RNA (58-MER)
    • Complex: DNA
      • DNA: DNA (39-MER)
      • DNA: DNA (5'-D(P*AP*GP*CP*AP*GP*CP*TP*GP*CP*AP*CP*C)-3')

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Supramolecule #1: Csy-AcrIF4-dsDNA

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

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Supramolecule #2: Csy-AcrIF4

SupramoleculeName: Csy-AcrIF4 / 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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Macromolecule #1: Type I-F CRISPR-associated protein Csy1

MacromoleculeName: Type I-F CRISPR-associated protein Csy1 / type: protein_or_peptide / ID: 1 / Details: WP_004343803.1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 49.313254 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTSPLPTPTW QELRQFIESF IQERLQGKLD KLHPDEDDKR QTLLATHRRE AWLADAARRV GQLQLVTHTL KPIHPDARGS NLHSLPQAP GQPGLAGSHE LGDRLVSDVV GNAAALDVFK FLSLQYQGKN LLNWLTEDSA EAVQALSDNA EQAREWRQAF I GITAVKGA ...String:
MTSPLPTPTW QELRQFIESF IQERLQGKLD KLHPDEDDKR QTLLATHRRE AWLADAARRV GQLQLVTHTL KPIHPDARGS NLHSLPQAP GQPGLAGSHE LGDRLVSDVV GNAAALDVFK FLSLQYQGKN LLNWLTEDSA EAVQALSDNA EQAREWRQAF I GITAVKGA PASHSLAKQL YFPLPGSGYH LLAPLFPTSL VHHVHALLRE ARFGDAAKAA REARSRQESW PHGFSEYPNL AI QKFGGTK PQNISQLNSE RYGENWLLPS LPPHWQRQDQ RAPIRHSSVF EHDFGRSPEV SRLTRTLQRL LAKTRHNNFT IRR YRAQLV GQICDEALQY AARLRELEPG WSATPGCQLH DAEQLWLDPL RAQTDETFLQ RRLRGDWPAE VGNRFANWLN RAVS SDSQI LGSPEAAQWS QELSKELTMF KEILEDERD

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Macromolecule #2: CRISPR-associated protein Csy3

MacromoleculeName: CRISPR-associated protein Csy3 / type: protein_or_peptide / ID: 2 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 37.623324 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSKPILSTAS VLAFERKLDP SDALMSAGAW AQRDASQEWP AVTVREKSVR GTISNRLKTK DRDPAKLDAS IQSPNLQTVD VANLPSDAD TLKVRFTLRV LGGAGTPSAC NDAAYRDKLL QTVATYVNEQ GFAELARRYA HNLANARFLW RNRVGAEAVE V RINHIRQG ...String:
MSKPILSTAS VLAFERKLDP SDALMSAGAW AQRDASQEWP AVTVREKSVR GTISNRLKTK DRDPAKLDAS IQSPNLQTVD VANLPSDAD TLKVRFTLRV LGGAGTPSAC NDAAYRDKLL QTVATYVNEQ GFAELARRYA HNLANARFLW RNRVGAEAVE V RINHIRQG EVARTWRFDA LAIGLRDFKA DAELDALAEL IASGLSGSGH VLLEVVAFAR IGDGQEVFPS QELILDKGDK KG QKSKTLY SVRDAAAIHS QKIGNALRTI DTWYPDEDGL GPIAVEPYGS VTSQGKAYRQ PKQKLDFYTL LDNWVLRDEA PAV EQQHYV IANLIRGGVF GEAEEK

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Macromolecule #3: CRISPR type I-F/YPEST-associated protein Csy2

MacromoleculeName: CRISPR type I-F/YPEST-associated protein Csy2 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 36.244074 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSVTDPEALL LLPRLSIQNA NAISSPLTWG FPSPGAFTGF VHALQRRVGI SLDIELDGVG IVCHRFEAQI SQPAGKRTKV FNLTRNPLN RDGSTAAIVE EGRAHLEVSL LLGVHGDGLD DHPAQEIARQ VQEQAGAMRL AGGSILPWCN ERFPAPNAEL L MLGGSDEQ ...String:
MSVTDPEALL LLPRLSIQNA NAISSPLTWG FPSPGAFTGF VHALQRRVGI SLDIELDGVG IVCHRFEAQI SQPAGKRTKV FNLTRNPLN RDGSTAAIVE EGRAHLEVSL LLGVHGDGLD DHPAQEIARQ VQEQAGAMRL AGGSILPWCN ERFPAPNAEL L MLGGSDEQ RRKNQRRLTR RLLPGFALVS REALLQQHLE TLRTTLPEAT TLDALLDLCR INFEPPATSS EEEASPPDAA WQ VRDKPGW LVPIPAGYNA LSPLYLPGEV RNARDRETPL RFVENLFGLG EWLSPHRVAA LSDLLWYHHA EPDKGLYRWS TPR FVEHAI A

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Macromolecule #4: AcrIF4

MacromoleculeName: AcrIF4 / type: protein_or_peptide / ID: 4 / Details: WP_016068584.1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 11.115526 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MMTISKTDID CYLQTYVVID PVSNGWQWGI DENGVGGALH HGRVEMVEGE NGYFGLRGAT HPTEKEAMAA ALGYLWRCRQ DLVAIARND AIEAEKYRAK A

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Macromolecule #5: Csy4

MacromoleculeName: Csy4 / type: protein_or_peptide / ID: 5 / Details: WP_004343806.1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 21.429477 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MDHYLDIRLR PDPEFPPAQL MSVLFGKLHQ ALVAQGGDRI GVSFPDLDES RSRLGERLRI HASADDLRAL LARPWLEGLR DHLQFGEPA VVPHPTPYRQ VSRVQAKSNP ERLRRRLMRR HDLSEEEARK RIPDTVARTL DLPFVTLRSQ STGQHFRLFI R HGPLQATA EEGGFTCYGL SKGGFVPWF

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Macromolecule #6: RNA (58-MER)

MacromoleculeName: RNA (58-MER) / type: rna / ID: 6 / Number of copies: 1
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 19.265404 KDa
SequenceString:
CUAAGAAAUU CACGGCGGGC UUGAUGUCCG CGUCUACCUG GUUCACUGCC GUGUAGGCAG

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Macromolecule #7: DNA (39-MER)

MacromoleculeName: DNA (39-MER) / type: dna / ID: 7 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 16.708691 KDa
SequenceString: (DG)(DG)(DA)(DA)(DG)(DC)(DC)(DA)(DT)(DC) (DC)(DA)(DG)(DG)(DT)(DA)(DG)(DA)(DC)(DG) (DC)(DG)(DG)(DA)(DC)(DA)(DT)(DC)(DA) (DA)(DG)(DC)(DC)(DC)(DG)(DC)(DC)(DG)(DT) (DG) (DA)(DA)(DG)(DG)(DT)(DG) ...String:
(DG)(DG)(DA)(DA)(DG)(DC)(DC)(DA)(DT)(DC) (DC)(DA)(DG)(DG)(DT)(DA)(DG)(DA)(DC)(DG) (DC)(DG)(DG)(DA)(DC)(DA)(DT)(DC)(DA) (DA)(DG)(DC)(DC)(DC)(DG)(DC)(DC)(DG)(DT) (DG) (DA)(DA)(DG)(DG)(DT)(DG)(DC)(DA) (DG)(DC)(DT)(DG)(DC)(DT)

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Macromolecule #8: DNA (5'-D(P*AP*GP*CP*AP*GP*CP*TP*GP*CP*AP*CP*C)-3')

MacromoleculeName: DNA (5'-D(P*AP*GP*CP*AP*GP*CP*TP*GP*CP*AP*CP*C)-3') / type: dna / ID: 8 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 16.574561 KDa
SequenceString: (DA)(DG)(DC)(DA)(DG)(DC)(DT)(DG)(DC)(DA) (DC)(DC)(DT)(DT)(DC)(DA)(DC)(DG)(DG)(DC) (DG)(DG)(DG)(DC)(DT)(DT)(DG)(DA)(DT) (DG)(DT)(DC)(DC)(DG)(DC)(DG)(DT)(DC)(DT) (DA) (DC)(DC)(DT)(DG)(DG)(DA) ...String:
(DA)(DG)(DC)(DA)(DG)(DC)(DT)(DG)(DC)(DA) (DC)(DC)(DT)(DT)(DC)(DA)(DC)(DG)(DG)(DC) (DG)(DG)(DG)(DC)(DT)(DT)(DG)(DA)(DT) (DG)(DT)(DC)(DC)(DG)(DC)(DG)(DT)(DC)(DT) (DA) (DC)(DC)(DT)(DG)(DG)(DA)(DT)(DG) (DG)(DC)(DT)(DT)(DC)(DC)

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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
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
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.37 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 117510
FSC plot (resolution estimation)

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