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- EMDB-31265: Structure of Csy-AcrIF5 -

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Basic information

Entry
Database: EMDB / ID: EMD-31265
TitleStructure of Csy-AcrIF5
Map data
Sample
  • Complex: Csy-AcrIF5
    • Complex: Csy-AcrIF5
      • Protein or peptide: CRISPR type I-F/YPEST-associated protein Csy2
      • Protein or peptide: CRISPR-associated protein Csy3
      • Protein or peptide: AcrIF5
      • Protein or peptide: type I-F CRISPR-associated endoribonuclease Cas6/Csy4
      • RNA: RNA (60-MER)
      • Protein or peptide: Type I-F CRISPR-associated protein Csy1
    • Complex: DNA
      • DNA: DNA (54-MER)
      • DNA: DNA (54-MER)
Keywordscomplex / 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
Uncharacterized protein / CRISPR-associated protein Csy3 / : / CRISPR type I-F/YPEST-associated protein Csy2
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsZhang L / Feng Y
Funding support China, 1 items
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, 2021-
Header (metadata) releaseMar 9, 2022-
Map releaseMar 9, 2022-
UpdateJun 5, 2024-
Current statusJun 5, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7eqg
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_31265.png

Downloads & links

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Map

FileDownload / File: emd_31265.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy AUTHOR: 0.0217 / Movie #1: 0.025
Minimum - Maximum-0.14626677 - 0.22332047
Average (Standard dev.)0.00044153573 (±0.006817533)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 264.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.11.11.1
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z264.000264.000264.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ330330330
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.1460.2230.000

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Supplemental data

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Sample components

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Entire : Csy-AcrIF5

EntireName: Csy-AcrIF5
Components
  • Complex: Csy-AcrIF5
    • Complex: Csy-AcrIF5
      • Protein or peptide: CRISPR type I-F/YPEST-associated protein Csy2
      • Protein or peptide: CRISPR-associated protein Csy3
      • Protein or peptide: AcrIF5
      • Protein or peptide: type I-F CRISPR-associated endoribonuclease Cas6/Csy4
      • RNA: RNA (60-MER)
      • Protein or peptide: Type I-F CRISPR-associated protein Csy1
    • Complex: DNA
      • DNA: DNA (54-MER)
      • DNA: DNA (54-MER)

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Supramolecule #1: Csy-AcrIF5

SupramoleculeName: Csy-AcrIF5 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#8

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Supramolecule #2: Csy-AcrIF5

SupramoleculeName: Csy-AcrIF5 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#5, #7
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)

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Supramolecule #3: DNA

SupramoleculeName: DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #6, #8

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Macromolecule #1: CRISPR type I-F/YPEST-associated protein Csy2

MacromoleculeName: CRISPR type I-F/YPEST-associated protein Csy2 / type: protein_or_peptide / ID: 1 / 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

UniProtKB: CRISPR type I-F/YPEST-associated protein Csy2

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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

UniProtKB: CRISPR-associated protein Csy3

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Macromolecule #3: AcrIF5

MacromoleculeName: AcrIF5 / type: protein_or_peptide / ID: 3 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 8.531865 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSRPTVVTVT ETPRNPGSYE VNVERDGKMV VGRARAGSDP GAAAAKAMQM AMEWGSPNYV ILGSNKVLAF IPEQLRVKM

UniProtKB: UNIPROTKB: A0A7M3A7R4

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Macromolecule #4: type I-F CRISPR-associated endoribonuclease Cas6/Csy4

MacromoleculeName: type I-F CRISPR-associated endoribonuclease Cas6/Csy4 / type: protein_or_peptide / ID: 4 / 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 #7: Type I-F CRISPR-associated protein Csy1

MacromoleculeName: Type I-F CRISPR-associated protein Csy1 / type: protein_or_peptide / ID: 7 / 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

UniProtKB: Uncharacterized protein

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Macromolecule #5: RNA (60-MER)

MacromoleculeName: RNA (60-MER) / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 19.265404 KDa
SequenceString:
CUAAGAAAUU CACGGCGGGC UUGAUGUCCG CGUCUACCUG GUUCACUGCC GUGUAGGCAG

GENBANK: GENBANK: HQ326201.1

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Macromolecule #6: DNA (54-MER)

MacromoleculeName: DNA (54-MER) / type: dna / ID: 6 / 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 (54-MER)

MacromoleculeName: DNA (54-MER) / 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: 7.5
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: FLOOD BEAM / Imaging mode: DARK FIELD
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 20.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
Initial angle assignmentType: OTHER
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 173096
FSC plot (resolution estimation)

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