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- EMDB-21517: Cryo-EM structure of anti-CRISPR AcrIF9 and dsDNA, bound to the t... -

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Basic information

Entry
Database: EMDB / ID: EMD-21517
TitleCryo-EM structure of anti-CRISPR AcrIF9 and dsDNA, bound to the type I-F crRNA-guided CRISPR surveillance complex
Map data
Sample
  • Complex: Anti-CRISPR AcrIF9 bound to the type I-F crRNA-guided CRISPR surveillance complex
    • Complex: type I-F crRNA-guided CRISPR surveillance complex
      • Protein or peptide: CRISPR-associated protein Csy1
      • Protein or peptide: Type I-F CRISPR-associated protein Csy2
      • Protein or peptide: CRISPR-associated protein Csy3
      • Protein or peptide: RNA (60-MER)
      • Protein or peptide: CRISPR-associated endonuclease Cas6/Csy4
      • Protein or peptide: non-target dsDNA
      • Protein or peptide: non-target dsDNA
    • Complex: Anti-CRISPR AcrIF9
      • Protein or peptide: anti-CRISPR AcrIF9
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 / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsHirschi M / Santiago-Frangos A / Wilkinson R / Golden SM / Wiedenheft B / Lander GC
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/Office of the DirectorDP2EB020402 United States
National Institutes of Health/Office of the DirectorS10OD021634 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM134867 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, 2020-
Header (metadata) releaseMay 13, 2020-
Map releaseMay 13, 2020-
UpdateJun 17, 2020-
Current statusJun 17, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6whi
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21517.png

Downloads & links

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Map

FileDownload / File: emd_21517.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.15 Å/pix.
x 192 pix.
= 220.8 Å		1.15 Å/pix.
x 192 pix.
= 220.8 Å		1.15 Å/pix.
x 192 pix.
= 220.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.15 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.012 / Movie #1: 0.012
Minimum - Maximum-0.05334784 - 0.07704133
Average (Standard dev.)0.00040360578 (±0.004277487)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 220.79999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.151.151.15
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z220.800220.800220.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-0.0530.0770.000

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Supplemental data

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Sample components

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Entire : Anti-CRISPR AcrIF9 bound to the type I-F crRNA-guided CRISPR surv...

EntireName: Anti-CRISPR AcrIF9 bound to the type I-F crRNA-guided CRISPR surveillance complex
Components
  • Complex: Anti-CRISPR AcrIF9 bound to the type I-F crRNA-guided CRISPR surveillance complex
    • Complex: type I-F crRNA-guided CRISPR surveillance complex
      • Protein or peptide: CRISPR-associated protein Csy1
      • Protein or peptide: Type I-F CRISPR-associated protein Csy2
      • Protein or peptide: CRISPR-associated protein Csy3
      • Protein or peptide: RNA (60-MER)
      • Protein or peptide: CRISPR-associated endonuclease Cas6/Csy4
      • Protein or peptide: non-target dsDNA
      • Protein or peptide: non-target dsDNA
    • Complex: Anti-CRISPR AcrIF9
      • Protein or peptide: anti-CRISPR AcrIF9

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Supramolecule #1: Anti-CRISPR AcrIF9 bound to the type I-F crRNA-guided CRISPR surv...

SupramoleculeName: Anti-CRISPR AcrIF9 bound to the type I-F crRNA-guided CRISPR surveillance complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: type I-F crRNA-guided CRISPR surveillance complex

SupramoleculeName: type I-F crRNA-guided CRISPR surveillance complex / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#5, #7-#8
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: Anti-CRISPR AcrIF9

SupramoleculeName: Anti-CRISPR AcrIF9 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #6
Source (natural)Organism: Proteus penneri (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: CRISPR-associated protein Csy1

MacromoleculeName: CRISPR-associated protein Csy1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
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: Type I-F CRISPR-associated protein Csy2

MacromoleculeName: Type I-F CRISPR-associated protein Csy2 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (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 protein Csy3

MacromoleculeName: CRISPR-associated protein Csy3 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (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 #4: RNA (60-MER)

MacromoleculeName: RNA (60-MER) / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
SequenceString:
CUAAGAAAUU CACGGCGGGC UUGAUGUCCG CGUCUACCUG GUUCACUGCC GUAUAGGCAG

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Macromolecule #5: CRISPR-associated endonuclease Cas6/Csy4

MacromoleculeName: CRISPR-associated endonuclease Cas6/Csy4 / type: protein_or_peptide / ID: 5 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (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 #6: anti-CRISPR AcrIF9

MacromoleculeName: anti-CRISPR AcrIF9 / type: protein_or_peptide / ID: 6 / Enantiomer: LEVO
Source (natural)Organism: Proteus penneri (bacteria)
SequenceString:
MKSTYIIKEV QNINSDREGV KVETTSLTSA KRIASKNQFF HGTVLRIESE SGNWLAYKED GKRWIECE

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Macromolecule #7: non-target dsDNA

MacromoleculeName: non-target dsDNA / type: protein_or_peptide / ID: 7 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
SequenceString: (DG)(DC)(DA)(DG)(DC)(DT)(DC)(DG)(DA)(DG) (DT)(DT)(DA)(DA)(DG)(DA)(DC)(DG)(DG)(DT) (DA)(DT)(DT)(DG)(DT)(DT)(DC)(DA)(DG) (DA)(DT)(DC)(DC)(DT)(DG)(DG)(DC)(DT)(DT) (DG) (DC)(DC)(DA)(DA)(DC)(DA) ...String:
(DG)(DC)(DA)(DG)(DC)(DT)(DC)(DG)(DA)(DG) (DT)(DT)(DA)(DA)(DG)(DA)(DC)(DG)(DG)(DT) (DA)(DT)(DT)(DG)(DT)(DT)(DC)(DA)(DG) (DA)(DT)(DC)(DC)(DT)(DG)(DG)(DC)(DT)(DT) (DG) (DC)(DC)(DA)(DA)(DC)(DA)(DG)(DT) (DG)(DA)(DT)(DT)(DT)(DG)(DC)(DT)(DC)(DA) (DT)(DT) (DT)(DT)(DG)(DT)(DA)(DG)(DA) (DT)(DT)(DG)(DA)(DG)(DT)(DC)(DG)(DC)(DT)

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Macromolecule #8: non-target dsDNA

MacromoleculeName: non-target dsDNA / type: protein_or_peptide / ID: 8 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
SequenceString: (DA)(DG)(DC)(DG)(DA)(DC)(DT)(DC)(DA)(DA) (DT)(DC)(DT)(DA)(DC)(DA)(DA)(DA)(DA)(DT) (DG)(DA)(DG)(DC)(DA)(DA)(DA)(DT)(DC) (DA)(DC)(DT)(DG)(DT)(DT)(DG)(DG)(DC)(DA) (DA) (DG)(DC)(DC)(DA)(DG)(DG) ...String:
(DA)(DG)(DC)(DG)(DA)(DC)(DT)(DC)(DA)(DA) (DT)(DC)(DT)(DA)(DC)(DA)(DA)(DA)(DA)(DT) (DG)(DA)(DG)(DC)(DA)(DA)(DA)(DT)(DC) (DA)(DC)(DT)(DG)(DT)(DT)(DG)(DG)(DC)(DA) (DA) (DG)(DC)(DC)(DA)(DG)(DG)(DA)(DT) (DC)(DT)(DG)(DA)(DA)(DC)(DA)(DA)(DT)(DA) (DC)(DC) (DG)(DT)(DC)(DT)(DT)(DA)(DA) (DC)(DT)(DC)(DG)(DA)(DG)(DC)(DT)(DG)(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
GridDetails: unspecified
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 6472 / Average exposure time: 11.5 sec. / Average electron dose: 66.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.2 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 36000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1049173
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 152066
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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