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- EMDB-41983: Structure of the phage immune evasion protein Gad1 bound to the G... -

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Basic information

Entry
Database: EMDB / ID: EMD-41983
TitleStructure of the phage immune evasion protein Gad1 bound to the Gabija GajAB complex
Map dataMain cryo-EM Map
Sample
  • Complex: Phage Phi3T Gad1 bound to the Bacillus cereus Gabija GajAB complex
    • Complex: Gabija GajAB complex
      • Protein or peptide: Endonuclease GajA
      • Protein or peptide: Gabija protein GajB
    • Complex: Gad1 Octamer
      • Protein or peptide: Gabija Anti-Defense 1
Keywordsviral immune evasion / phage / bacteria / anti-phage defense complex / DNA nuclease / DNA helicase / VIRAL PROTEIN
Function / homology
Function and homology information


DNA helicase activity / defense response to virus / endonuclease activity / Hydrolases; Acting on ester bonds / hydrolase activity / DNA binding / ATP binding / metal ion binding
Similarity search - Function
Overcoming lysogenization defect protein-like, TOPRIM domain / OLD protein-like, TOPRIM domain / AAA domain, group 15 / AAA ATPase domain / AAA domain / UvrD-like helicase C-terminal domain / UvrD-like helicase C-terminal domain / UvrD-like helicase, ATP-binding domain / UvrD-like DNA helicase ATP-binding domain profile. / DNA helicase, UvrD/REP type / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Transposase / Endonuclease GajA / Gabija protein GajB
Similarity search - Component
Biological speciesBacillus phage phi3T (virus) / Bacillus cereus VD045 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.57 Å
AuthorsAntine SP / Johnson AG / Mooney SE / Mayer ML / Kranzsuch PJ
Funding support United States, 5 items
OrganizationGrant numberCountry
The Pew Charitable Trusts United States
Burroughs Wellcome Fund United States
The G. Harold and Leila Y. Mathers Foundation United States
The Mark Foundation United States
Parker Institute for Cancer Immunotherapy United States
Citation
Journal: Nature / Year: 2024
Title: Structural basis of Gabija anti-phage defence and viral immune evasion.
Authors: Sadie P Antine / Alex G Johnson / Sarah E Mooney / Azita Leavitt / Megan L Mayer / Erez Yirmiya / Gil Amitai / Rotem Sorek / Philip J Kranzusch /
Abstract: Bacteria encode hundreds of diverse defence systems that protect them from viral infection and inhibit phage propagation. Gabija is one of the most prevalent anti-phage defence systems, occurring in ...Bacteria encode hundreds of diverse defence systems that protect them from viral infection and inhibit phage propagation. Gabija is one of the most prevalent anti-phage defence systems, occurring in more than 15% of all sequenced bacterial and archaeal genomes, but the molecular basis of how Gabija defends cells from viral infection remains poorly understood. Here we use X-ray crystallography and cryo-electron microscopy (cryo-EM) to define how Gabija proteins assemble into a supramolecular complex of around 500 kDa that degrades phage DNA. Gabija protein A (GajA) is a DNA endonuclease that tetramerizes to form the core of the anti-phage defence complex. Two sets of Gabija protein B (GajB) dimers dock at opposite sides of the complex and create a 4:4 GajA-GajB assembly (hereafter, GajAB) that is essential for phage resistance in vivo. We show that a phage-encoded protein, Gabija anti-defence 1 (Gad1), directly binds to the Gabija GajAB complex and inactivates defence. A cryo-EM structure of the virally inhibited state shows that Gad1 forms an octameric web that encases the GajAB complex and inhibits DNA recognition and cleavage. Our results reveal the structural basis of assembly of the Gabija anti-phage defence complex and define a unique mechanism of viral immune evasion.
#1: Journal: To Be Published
Title: Phages overcome bacterial immunity via diverse anti-defense proteins
Authors: Yirmiya E / Leavitt A / Lu A / Avraham C / Osterman I / Garb J / Antine SP / Mooney SE / Hobbs SJ / Kranzusch PJ / Amitai G / Sorek R
History
DepositionSep 15, 2023-
Header (metadata) releaseNov 22, 2023-
Map releaseNov 22, 2023-
UpdateJan 24, 2024-
Current statusJan 24, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41983.png

Downloads & links

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Map

FileDownload / File: emd_41983.map.gz / Format: CCP4 / Size: 163.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMain cryo-EM Map
Voxel sizeX=Y=Z: 0.89 Å
Density
Contour LevelBy AUTHOR: 0.028
Minimum - Maximum-0.095440485 - 0.268814
Average (Standard dev.)0.00024269697 (±0.00942611)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions350350350
Spacing350350350
CellA=B=C: 311.5 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map B

Fileemd_41983_half_map_1.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map A

Fileemd_41983_half_map_2.map
AnnotationHalf Map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Phage Phi3T Gad1 bound to the Bacillus cereus Gabija GajAB complex

EntireName: Phage Phi3T Gad1 bound to the Bacillus cereus Gabija GajAB complex
Components
  • Complex: Phage Phi3T Gad1 bound to the Bacillus cereus Gabija GajAB complex
    • Complex: Gabija GajAB complex
      • Protein or peptide: Endonuclease GajA
      • Protein or peptide: Gabija protein GajB
    • Complex: Gad1 Octamer
      • Protein or peptide: Gabija Anti-Defense 1

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Supramolecule #1: Phage Phi3T Gad1 bound to the Bacillus cereus Gabija GajAB complex

SupramoleculeName: Phage Phi3T Gad1 bound to the Bacillus cereus Gabija GajAB complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Bacillus phage phi3T (virus)
Molecular weightTheoretical: 280 KDa

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Supramolecule #2: Gabija GajAB complex

SupramoleculeName: Gabija GajAB complex / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Bacillus cereus VD045 (bacteria)

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Supramolecule #3: Gad1 Octamer

SupramoleculeName: Gad1 Octamer / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Bacillus phage phi3T (virus)

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Macromolecule #1: Endonuclease GajA

MacromoleculeName: Endonuclease GajA / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Bacillus cereus VD045 (bacteria)
Molecular weightTheoretical: 78.045656 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGSSHHHHHH GSGVKTENND HINLKVAGQD GSVVQFKIKR HTPLSKLMKA YCERQGLSMR QIRFRFDGQP INETDTPAQL EMEDEDTID VFQQQTGGSK FSNITIKNFR NFEKVNINLD NKNVIFGMND IGKTNFLYAL RFLLDKEIRK FGFNKSDYHK H DTSKKIEI ...String:
MGSSHHHHHH GSGVKTENND HINLKVAGQD GSVVQFKIKR HTPLSKLMKA YCERQGLSMR QIRFRFDGQP INETDTPAQL EMEDEDTID VFQQQTGGSK FSNITIKNFR NFEKVNINLD NKNVIFGMND IGKTNFLYAL RFLLDKEIRK FGFNKSDYHK H DTSKKIEI ILTLDLSNYE KDEDTKKLIS VVKGARTSAN ADVFYIALES KYDDKELYGN IILKWGSELD NLIDIPGRGN IN ALDNVFK VIYINPLVDL DKLFAQNKKY IFEESQGNES DEGILNNIKS LTDQVNQQIG EMTIIKGFQQ EITSEYRSLK KEE VSIELK SEMAIKGFFS DIIPYIKKDG DSNYYPTSGD GRRKMLSYSI YNYLAKKKYE DKIVIYLIEE PEISLHRSMQ IALS KQLFE QSTYKYFFLS THSPELLYEM DNTRLIRVHS TEKVVCSSHM YNVEEAYGSV KKKLNKALSS ALFAERVLLI EGPSE KILF EKVLDEVEPE YELNGGFLLE VGGTYFNHYV CTLNDLGITH IIKTDNDLKS KKGKKGVYEL LGLNRCLNLL GRENLD EIT IDIPEDIKGK KKKERLNERK KEIFKQYKNE VGEFLGERIY LSEIDLENDL YSAIGESMKR IFENEDPVHY LQKSKLF NM VELVNNLSTK DCFDVFEHEK FACLKELVGS DRG

UniProtKB: Endonuclease GajA

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Macromolecule #2: Gabija protein GajB

MacromoleculeName: Gabija protein GajB / type: protein_or_peptide / ID: 2 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Bacillus cereus VD045 (bacteria)
Molecular weightTheoretical: 57.139992 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSREQIIKDG GNILVTAGAG SGKTTILVSK IEADLKENKT HYSIAAVTFT NKAAKEIEGR LGYSSRGNFI GTNDGFVESE IIRPFIKDA FGNDYPDNFT AEYFDNQFAS YDKGLQVLKY QNILGTYSNP KKNFKFQLAL DILKKSLVAR QYIFSKYFKI F IDEYQDSD ...String:
MSREQIIKDG GNILVTAGAG SGKTTILVSK IEADLKENKT HYSIAAVTFT NKAAKEIEGR LGYSSRGNFI GTNDGFVESE IIRPFIKDA FGNDYPDNFT AEYFDNQFAS YDKGLQVLKY QNILGTYSNP KKNFKFQLAL DILKKSLVAR QYIFSKYFKI F IDEYQDSD KDMHNLFMYL KDQLKIKLFI VGDPKQSIYI WRGAEPENFN GLIENSTDFN KYHLTSNFRC CQDIQNYSNL FN EETRSLI KEKNEVQNVI SIADDMPISD ILLKLTEEKQ VLNIEAELVI LVRRRNQAIE IMKELNEEGF NFIFIPQTPL DRA TPNATL LKEVIKYVKN DRYSIYDLAA EIVGNLSSRE IKEIQKIINE LLVPNINQVL INQVLINLFA KLEITLDTRE ITAF TEVMM TNEFDIAFDT NEYLHKIFTV HSAKGLEFNQ VIITASDYNV HYNRDTNEHY VATTRAKDKL IVIMDNKKYS DYIET LMKE LKIKNIIKSI

UniProtKB: Gabija protein GajB

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Macromolecule #3: Gabija Anti-Defense 1

MacromoleculeName: Gabija Anti-Defense 1 / type: protein_or_peptide / ID: 3 / Number of copies: 8 / Enantiomer: LEVO
Source (natural)Organism: Bacillus phage phi3T (virus)
Molecular weightTheoretical: 34.919184 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MKLIGIKTSN CFLVSDNIEG KRYFHSQLDE LLFDGKRATE TYKSDWFKLE KEPSVIEKQM PAKKINHRYE LKEGFQESEL TPKVIKASY IGEDSEYYEV KGLYDLKFEE IPQQNEKIEF EMNVIEEIDG ELKLQSHNFN LNYNLLDRIQ THPMLLETKP C YLSQEESY ...String:
MKLIGIKTSN CFLVSDNIEG KRYFHSQLDE LLFDGKRATE TYKSDWFKLE KEPSVIEKQM PAKKINHRYE LKEGFQESEL TPKVIKASY IGEDSEYYEV KGLYDLKFEE IPQQNEKIEF EMNVIEEIDG ELKLQSHNFN LNYNLLDRIQ THPMLLETKP C YLSQEESY KIIRNHIKAN INPKFARITS DYDFCLTVVK VLELYKPHEY IVDLNAMYKR RKPKLEKRFQ TKREVEIYKV AP KAYQSYP IVEPFSGKDV EDLKSNIKKF LDDLMAKINE PLVECKCCKG RGVILNEN

UniProtKB: Transposase

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation state3D array

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Sample preparation

Concentration1.00 mg/mL
BufferpH: 7.5
Details: 20 mM HEPES-KOH pH 7.5, 20 mM KCl, and 1 mM TCEP-KOH
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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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: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.9000000000000001 µm / Nominal defocus min: 0.8 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 41.1 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.57 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 351193
FSC plot (resolution estimation)

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