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- EMDB-25789: Cryo-EM structure of the Csy-AcrIF24-promoter DNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-25789
TitleCryo-EM structure of the Csy-AcrIF24-promoter DNA complex
Map data
Sample
  • Complex: Csy-AcrIF
    • Protein or peptide: CRISPR-associated protein Csy1
    • Protein or peptide: CRISPR type I-F/YPEST-associated protein Csy2
    • Protein or peptide: CRISPR-associated endonuclease Cas6/Csy4
    • Protein or peptide: CRISPR type I-F/YPEST-associated protein Csy3
    • Protein or peptide: AcrIF24
    • RNA: RNA (61-MER)
    • DNA: DNA (5'-D(P*AP*TP*AP*GP*CP*TP*CP*GP*AP*TP*TP*CP*GP*AP*GP*CP*TP*AP*A)-3')
    • DNA: DNA (5'-D(P*TP*TP*AP*GP*CP*TP*CP*GP*AP*AP*TP*CP*GP*AP*GP*CP*TP*AP*T)-3')
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
CRISPR type I-F/YPEST-associated protein Csy3 / CRISPR type I-F/YPEST-associated protein Csy2 / CRISPR-associated protein Csy1 / CRISPR-associated endonuclease Cas6/Csy4
Similarity search - Component
Biological speciesPseudomonas (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsMukherjee IA / Chang L
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM138675 United States
CitationJournal: Nat Chem Biol / Year: 2022
Title: Structural basis of AcrIF24 as an anti-CRISPR protein and transcriptional suppressor.
Authors: Indranil Arun Mukherjee / Clinton Gabel / Nicholas Noinaj / Joseph Bondy-Denomy / Leifu Chang /
Abstract: Anti-CRISPR (Acr) proteins are encoded by phages to inactivate CRISPR-Cas systems of bacteria and archaea and are used to enhance the CRISPR toolbox for genome editing. Here we report the structure ...Anti-CRISPR (Acr) proteins are encoded by phages to inactivate CRISPR-Cas systems of bacteria and archaea and are used to enhance the CRISPR toolbox for genome editing. Here we report the structure and mechanism of AcrIF24, an Acr protein that inhibits the type I-F CRISPR-Cas system from Pseudomonas aeruginosa. AcrIF24 is a homodimer that associates with two copies of the surveillance complex (Csy) and prevents the hybridization between CRISPR RNA and target DNA. Furthermore, AcrIF24 functions as an anti-CRISPR-associated (Aca) protein to repress the transcription of the acrIF23-acrIF24 operon. Alone or in complex with Csy, AcrIF24 is capable of binding to the acrIF23-acrIF24 promoter DNA with nanomolar affinity. The structure of a Csy-AcrIF24-promoter DNA complex at 2.7 Å reveals the mechanism for transcriptional suppression. Our results reveal that AcrIF24 functions as an Acr-Aca fusion protein, and they extend understanding of the diverse mechanisms used by Acr proteins.
History
DepositionDec 21, 2021-
Header (metadata) releaseSep 21, 2022-
Map releaseSep 21, 2022-
UpdateDec 7, 2022-
Current statusDec 7, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25789.png

Downloads & links

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Map

FileDownload / File: emd_25789.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.05 Å
Density
Contour LevelBy AUTHOR: 0.08
Minimum - Maximum-0.51651186 - 1.2403067
Average (Standard dev.)-0.00021653452 (±0.026753642)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 336.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Csy-AcrIF

EntireName: Csy-AcrIF
Components
  • Complex: Csy-AcrIF
    • Protein or peptide: CRISPR-associated protein Csy1
    • Protein or peptide: CRISPR type I-F/YPEST-associated protein Csy2
    • Protein or peptide: CRISPR-associated endonuclease Cas6/Csy4
    • Protein or peptide: CRISPR type I-F/YPEST-associated protein Csy3
    • Protein or peptide: AcrIF24
    • RNA: RNA (61-MER)
    • DNA: DNA (5'-D(P*AP*TP*AP*GP*CP*TP*CP*GP*AP*TP*TP*CP*GP*AP*GP*CP*TP*AP*A)-3')
    • DNA: DNA (5'-D(P*TP*TP*AP*GP*CP*TP*CP*GP*AP*AP*TP*CP*GP*AP*GP*CP*TP*AP*T)-3')

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Supramolecule #1: Csy-AcrIF

SupramoleculeName: Csy-AcrIF / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Pseudomonas (bacteria)
Molecular weightTheoretical: 400 kDa/nm

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Macromolecule #1: CRISPR-associated protein Csy1

MacromoleculeName: CRISPR-associated protein Csy1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas (bacteria)
Molecular weightTheoretical: 49.194168 KDa
Recombinant expressionOrganism: Escherichia (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 EHDFGRTPEV SRLTRTLQRF LAKTVHNNLA IRQ RRAQLV AQICDEALQY AARLRELEPG WSATPGCQLH DAEQLWLDPL RAQTDETFLQ RRLRGDWPAE VGNRFANWLN RAVS SDSQI LGSPEAAQWS QELSKELTMF KEILEDERD

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Macromolecule #2: CRISPR type I-F/YPEST-associated protein Csy2

MacromoleculeName: CRISPR type I-F/YPEST-associated protein Csy2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas (bacteria)
Molecular weightTheoretical: 36.244074 KDa
Recombinant expressionOrganism: Escherichia (bacteria)
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 #3: CRISPR-associated endonuclease Cas6/Csy4

MacromoleculeName: CRISPR-associated endonuclease Cas6/Csy4 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Pseudomonas (bacteria)
Molecular weightTheoretical: 21.427504 KDa
Recombinant expressionOrganism: Escherichia (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 #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 (bacteria)
Molecular weightTheoretical: 39.778594 KDa
Recombinant expressionOrganism: Escherichia (bacteria)
SequenceString: MKSSHHHHHH ENLYFQSNAS KPILSTASVL AFERKLDPSD ALMSAGAWAQ RDASQEWPAV TVREKSVRGT ISNRLKTKDR DPAKLDASI QSPNLQTVDV ANLPSDADTL KVRFTLRVLG GAGTPSACND AAYRDKLLQT VATYVNDQGF AELARRYAHN L ANARFLWR ...String:
MKSSHHHHHH ENLYFQSNAS KPILSTASVL AFERKLDPSD ALMSAGAWAQ RDASQEWPAV TVREKSVRGT ISNRLKTKDR DPAKLDASI QSPNLQTVDV ANLPSDADTL KVRFTLRVLG GAGTPSACND AAYRDKLLQT VATYVNDQGF AELARRYAHN L ANARFLWR NRVGAEAVEV RINHIRQGEV ARAWRFDALA IGLRDFKADA ELDALAELIA SGLSGSGHVL LEVVAFARIG DG QEVFPSQ ELILDKGDKK GQKSKTLYSV RDAAAIHSQK IGNALRTIDT WYPDEDGLGP IAVEPYGSVT SQGKAYRQPK QKL DFYTLL DNWVLRDEAP AVEQQHYVIA NLIRGGVFGE AEEK

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Macromolecule #5: AcrIF24

MacromoleculeName: AcrIF24 / type: protein_or_peptide / ID: 5 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas (bacteria)
Molecular weightTheoretical: 24.995271 KDa
Recombinant expressionOrganism: Escherichia (bacteria)
SequenceString: MNAIHIGPFS ITPAARGLHY GGLPHHQWTL YYGPREMAIK TLPDSYTSSE VRDEFSDIIA EFVIDARHRY APDVLELVNS DGDAVLARV AVSRLPEALS GCIPDDRFPY WLLTASRPRL GLPVTLNEYT ALAVELSAPP LAWITGLLPG EVLTHDAEEW R PPTSWELR ...String:
MNAIHIGPFS ITPAARGLHY GGLPHHQWTL YYGPREMAIK TLPDSYTSSE VRDEFSDIIA EFVIDARHRY APDVLELVNS DGDAVLARV AVSRLPEALS GCIPDDRFPY WLLTASRPRL GLPVTLNEYT ALAVELSAPP LAWITGLLPG EVLTHDAEEW R PPTSWELR HVVGEGSFTG VSGAAAAALL GMSATNFRKY TAGDSAANRQ KISFAAWHYL LDRLGVKRAS

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Macromolecule #6: RNA (61-MER)

MacromoleculeName: RNA (61-MER) / type: rna / ID: 6 / Number of copies: 1
Source (natural)Organism: Pseudomonas (bacteria)
Molecular weightTheoretical: 19.538586 KDa
SequenceString:
CUAAGAAAUU CACGGCGGGC UUGAUGUCCG CGUCUACCUG AUUCACUGCC GUAUAGGCAG C

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Macromolecule #7: DNA (5'-D(P*AP*TP*AP*GP*CP*TP*CP*GP*AP*TP*TP*CP*GP*AP*GP*CP*TP*AP...

MacromoleculeName: DNA (5'-D(P*AP*TP*AP*GP*CP*TP*CP*GP*AP*TP*TP*CP*GP*AP*GP*CP*TP*AP*A)-3')
type: dna / ID: 7 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Pseudomonas (bacteria)
Molecular weightTheoretical: 5.828798 KDa
SequenceString:
(DA)(DT)(DA)(DG)(DC)(DT)(DC)(DG)(DA)(DT) (DT)(DC)(DG)(DA)(DG)(DC)(DT)(DA)(DA)

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

MacromoleculeName: DNA (5'-D(P*TP*TP*AP*GP*CP*TP*CP*GP*AP*AP*TP*CP*GP*AP*GP*CP*TP*AP*T)-3')
type: dna / ID: 8 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Pseudomonas (bacteria)
Molecular weightTheoretical: 5.819784 KDa
SequenceString:
(DT)(DT)(DA)(DG)(DC)(DT)(DC)(DG)(DA)(DA) (DT)(DC)(DG)(DA)(DG)(DC)(DT)(DA)(DT)

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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

Concentration3.5 mg/mL
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: OTHER / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (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: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 426924

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Atomic model buiding 1

Initial modelPDB ID:

7jzw

RefinementSpace: REAL
Output model

PDB-7tax:
Cryo-EM structure of the Csy-AcrIF24-promoter DNA complex

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