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- EMDB-21516: Cryo-EM structure of anti-CRISPR AcrIF9, bound to the type I-F cr... -

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Basic information

Entry
Database: EMDB / ID: EMD-21516
TitleCryo-EM structure of anti-CRISPR AcrIF9, bound to the type I-F crRNA-guided CRISPR surveillance complex
Map dataanti-CRISPR AcrIF9 bound to the type I-F crRNA-guided CRISPR surveillance complex
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
      • RNA: RNA (60-MER)
      • Protein or peptide: CRISPR-associated endonuclease Cas6/Csy4
    • Complex: Anti-CRISPR AcrIF9
      • Protein or peptide: anti-CRISPR AcrIF9
Keywordstype I-F CRISPR RNA-guided surveillance complex / Csy complex / IMMUNE SYSTEM-RNA complex
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: 3.9 Å
AuthorsHirschi M / Santiago-Frangos A
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/Office of the DirectorDP2EB020402 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM134867 United States
National Institutes of Health/Office of the DirectorS10OD021634 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-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6w1x
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21516.png

Downloads & links

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Map

FileDownload / File: emd_21516.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationanti-CRISPR AcrIF9 bound to the type I-F crRNA-guided CRISPR surveillance complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.15 Å/pix.
x 200 pix.
= 230. Å		1.15 Å/pix.
x 200 pix.
= 230. Å		1.15 Å/pix.
x 200 pix.
= 230. 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: 0.015 / Movie #1: 0.015
Minimum - Maximum-0.061401043 - 0.11653317
Average (Standard dev.)-0.000022094737 (±0.004716737)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 230.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.151.151.15
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z230.000230.000230.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0610.117-0.000

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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
      • RNA: RNA (60-MER)
      • Protein or peptide: CRISPR-associated endonuclease Cas6/Csy4
    • 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: #1-#6

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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
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)

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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)

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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 aeruginosa (bacteria)
Molecular weightTheoretical: 49.194168 KDa
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

UniProtKB: CRISPR-associated protein Csy1

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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 / 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: Uncharacterized protein

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Macromolecule #3: CRISPR-associated protein Csy3

MacromoleculeName: CRISPR-associated protein Csy3 / type: protein_or_peptide / ID: 3 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 39.778594 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
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

UniProtKB: CRISPR type I-F/YPEST-associated protein Csy3

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Macromolecule #5: CRISPR-associated endonuclease Cas6/Csy4

MacromoleculeName: CRISPR-associated endonuclease Cas6/Csy4 / type: protein_or_peptide / ID: 5 / 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 (E. coli)
SequenceString:
MDHYLDIRLR PDPEFPPAQL MSVLFGKLHQ ALVAQGGDRI GVSFPDLDES RSRLGERLRI HASADDLRAL LARPWLEGLR DHLQFGEPA VVPHPTPYRQ VSRVQAKSNP ERLRRRLMRR HDLSEEEARK RIPDTVARAL DLPFVTLRSQ STGQHFRLFI R HGPLQVTA EEGGFTCYGL SKGGFVPWF

UniProtKB: CRISPR-associated endonuclease Cas6/Csy4

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Macromolecule #6: anti-CRISPR AcrIF9

MacromoleculeName: anti-CRISPR AcrIF9 / type: protein_or_peptide / ID: 6 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Proteus penneri (bacteria)
Molecular weightTheoretical: 7.863849 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MKSTYIIKEV QNINSDREGV KVETTSLTSA KRIASKNQFF HGTVLRIESE SGNWLAYKED GKRWIECE

UniProtKB: SH3b domain-containing protein

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Macromolecule #4: RNA (60-MER)

MacromoleculeName: RNA (60-MER) / type: rna / ID: 4 / 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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormula
20.0 mMHEPES
100.0 mMNaCl
2.5 %glycerol
1.0 mMTCEP
GridModel: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 7 sec. / Pretreatment - Atmosphere: OTHER / Details: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: HOMEMADE PLUNGER

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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: 1653 / Average exposure time: 13.2 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: 1571636
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 285707
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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