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- EMDB-22582: Cryo-EM structure of CRISPR-Cas surveillance complex with AcrIF4 -

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Basic information

Entry
Database: EMDB / ID: EMD-22582
TitleCryo-EM structure of CRISPR-Cas surveillance complex with AcrIF4
Map data
Sample
  • Complex: CRISPR-Cas complexCRISPR
    • Protein or peptide: CRISPR type I-F/YPEST-associated protein Csy1
    • Protein or peptide: CRISPR-associated endonuclease Cas6/Csy4
    • Protein or peptide: CRISPR type I-F/YPEST-associated protein Csy2
    • Protein or peptide: CRISPR type I-F/YPEST-associated protein Csy3
    • Protein or peptide: Type I-F anti-CRISPR protein
    • RNA: CRISPR repeat sequence
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
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)
Methodsingle 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, 2020-
Header (metadata) releaseDec 30, 2020-
Map releaseDec 30, 2020-
UpdateJan 12, 2022-
Current statusJan 12, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0344
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0344
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7jzw
  • Surface level: 0.0344
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22582.png

Downloads & links

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Map

FileDownload / File: emd_22582.map.gz / Format: CCP4 / Size: 65.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.05 Å
Density
Contour LevelBy AUTHOR: 0.0344 / Movie #1: 0.0344
Minimum - Maximum-0.13030991 - 0.26355678
Average (Standard dev.)0.0006691232 (±0.006441125)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions258258258
Spacing258258258
CellA=B=C: 270.9 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.051.051.05
M x/y/z258258258
origin x/y/z0.0000.0000.000
length x/y/z270.900270.900270.900
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS258258258
D min/max/mean-0.1300.2640.001

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Supplemental data

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Sample components

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Entire : CRISPR-Cas complex

EntireName: CRISPR-Cas complexCRISPR
Components
  • Complex: CRISPR-Cas complexCRISPR
    • Protein or peptide: CRISPR type I-F/YPEST-associated protein Csy1
    • Protein or peptide: CRISPR-associated endonuclease Cas6/Csy4
    • Protein or peptide: CRISPR type I-F/YPEST-associated protein Csy2
    • Protein or peptide: CRISPR type I-F/YPEST-associated protein Csy3
    • Protein or peptide: Type I-F anti-CRISPR protein
    • RNA: CRISPR repeat sequence

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Supramolecule #1: CRISPR-Cas complex

SupramoleculeName: CRISPR-Cas complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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

MacromoleculeName: CRISPR type I-F/YPEST-associated protein Csy1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 49.136133 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MTSPLPTPTW QELRQFIESF IQERLQGKLD KLQPDEDDKR QTLLATHRRE AWLADAARRV GQLQLVTHTL KPIHPDARGS NLHSLPQAP GQPGLAGSHE LGDRLVSDVV GNAAALDVFK FLSLQYQGKN LLNWLTEDSA EALQALSDNA EQAREWRQAF I GITTVKGA ...String:
MTSPLPTPTW QELRQFIESF IQERLQGKLD KLQPDEDDKR QTLLATHRRE AWLADAARRV GQLQLVTHTL KPIHPDARGS NLHSLPQAP GQPGLAGSHE LGDRLVSDVV GNAAALDVFK FLSLQYQGKN LLNWLTEDSA EALQALSDNA EQAREWRQAF I GITTVKGA PASHSLAKQL YFPLPGSGYH LLAPLFPTSL VHHVHALLRE ARFGDAAKAA REARSRQESW PHGFSEYPNL AI QKFGGTK PQNISQLNNE RRGENWLLPS LPPNWQRQNV NAPMRHSSVF AHDFGRTPEV SRLTRTLQRF LAKTVHNNLA IRQ RRAQLV AQICDEALQY AARLRELEPG WSATPGCQLH DAEQLWLDPL RAQTDETFLQ RRLRGDWPAE VGNRFANWLN RAVS SDSQI LGSPEAAQWS QELSKELTMF KEILEDERD

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Macromolecule #2: CRISPR-associated endonuclease Cas6/Csy4

MacromoleculeName: CRISPR-associated endonuclease Cas6/Csy4 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 21.427504 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MDHYLDIRLR PDPEFPPAQL MSVLFGKLHQ ALVAQGGDRI GVSFPDLDES RSRLGERLRI HASADDLRAL LARPWLEGLR DHLQFGEPA VVPHPTPYRQ VSRVQAKSNP ERLRRRLMRR HDLSEEEARK RIPDTVARAL DLPFVTLRSQ STGQHFRLFI R HGPLQVTA EEGGFTCYGL SKGGFVPWF

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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: 36.446352 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MAMSVTDPEA LLLLPRLSIQ NANAISSPLT WGFPSPGAFT GFVHALQRRV GISLDIELDG VGIVCHRFEA QISQPAGKRT KVFNLTRNP LNRDGSTAAI VEEGRAHLEV SLLLGVHGDG LDDHPAQEIA RQVQEQAGAM RLAGGSILPW CNERFPAPNA E LLMLGGSD ...String:
MAMSVTDPEA LLLLPRLSIQ NANAISSPLT WGFPSPGAFT GFVHALQRRV GISLDIELDG VGIVCHRFEA QISQPAGKRT KVFNLTRNP LNRDGSTAAI VEEGRAHLEV SLLLGVHGDG LDDHPAQEIA RQVQEQAGAM RLAGGSILPW CNERFPAPNA E LLMLGGSD EQRRKNQRRL TRRLLPGFAL VSREALLQQH LETLRTTLPE ATTLDALLDL CRINFEPPAT SSEEEASPPD AA WQVRDKP GWLVPIPAGY NALSPLYLPG EVRNARDRET PLRFVENLFG LGEWLSPHRV AALSDLLWYH HAEPDKGLYR WST PRFVEH AIA

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Macromolecule #4: CRISPR type I-F/YPEST-associated protein Csy3

MacromoleculeName: CRISPR type I-F/YPEST-associated protein Csy3 / type: protein_or_peptide / ID: 4 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 37.781547 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MAMSKPILST ASVLAFERKL DPSDALMSAG AWAQRDASQE WPAVTVREKS VRGTISNRLK TKDRDPAKLD ASIQSPNLQT VDVANLPSD ADTLKVRFTL RVLGGAGTPS ACNDAAYRDK LLQTVATYVN DQGFAELARR YAHNLANARF LWRNRVGAEA V EVRINHIR ...String:
MAMSKPILST ASVLAFERKL DPSDALMSAG AWAQRDASQE WPAVTVREKS VRGTISNRLK TKDRDPAKLD ASIQSPNLQT VDVANLPSD ADTLKVRFTL RVLGGAGTPS ACNDAAYRDK LLQTVATYVN DQGFAELARR YAHNLANARF LWRNRVGAEA V EVRINHIR QGEVARAWRF DALAIGLRDF KADAELDALA ELIASGLSGS GHVLLEVVAF ARIGDGQEVF PSQELILDKG DK KGQKSKT LYSVRDAAAI HSQKIGNALR TIDTWYPDED GLGPIAVEPY GSVTSQGKAY RQPKQKLDFY TLLDNWVLRD EAP AVEQQH YVIANLIRGG VFGEAEEK

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Macromolecule #5: Type I-F anti-CRISPR protein

MacromoleculeName: Type I-F anti-CRISPR protein / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas phage sp. (virus)
Molecular weightTheoretical: 11.087513 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MMTISKTDID CYLQTYVVID PVSNGWQWGI DENGVGGALH HGRVEMVEGE NGYFGLRGAT HPTEKEAMAA ALGYLWKCRQ DLVAIARND AIEAEKYRAK A

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Macromolecule #6: CRISPR repeat sequence

MacromoleculeName: CRISPR repeat sequence / type: rna / ID: 6 / Number of copies: 1
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 19.538586 KDa
SequenceString:
CUAAGAAAUU CACGGCGGGC UUGAUGUCCG CGUCUACCUG AUUCACUGCC GUAUAGGCAG C

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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: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 54.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 766782

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