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- PDB-8u7i: Structure of the phage immune evasion protein Gad1 bound to the G... -

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Basic information

Entry
Database: PDB / ID: 8u7i
TitleStructure of the phage immune evasion protein Gad1 bound to the Gabija GajAB complex
Components
  • Endonuclease GajA
  • Gabija Anti-Defense 1
  • Gabija protein GajB
KeywordsVIRAL PROTEIN / viral immune evasion / phage / bacteria / anti-phage defense complex / DNA nuclease / DNA helicase
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 cereus VD045 (bacteria)
Bacillus phage phi3T (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.57 Å
AuthorsAntine, S.P. / Johnson, A.G. / Mooney, S.E. / Mayer, M.L. / Kranzsuch, P.J.
Funding support United States, 5items
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, S.P. / Mooney, S.E. / Hobbs, S.J. / Kranzusch, P.J. / Amitai, G. / Sorek, R.
History
DepositionSep 15, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 22, 2023Provider: repository / Type: Initial release
Revision 1.1Dec 6, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Jan 24, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Endonuclease GajA
B: Endonuclease GajA
C: Endonuclease GajA
D: Endonuclease GajA
E: Gabija protein GajB
F: Gabija protein GajB
G: Gabija protein GajB
H: Gabija protein GajB
I: Gabija Anti-Defense 1
J: Gabija Anti-Defense 1
K: Gabija Anti-Defense 1
L: Gabija Anti-Defense 1
M: Gabija Anti-Defense 1
N: Gabija Anti-Defense 1
O: Gabija Anti-Defense 1
P: Gabija Anti-Defense 1


Theoretical massNumber of molelcules
Total (without water)820,09616
Polymers820,09616
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Endonuclease GajA


Mass: 78045.656 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus cereus VD045 (bacteria) / Gene: gajA / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: J8H9C1
#2: Protein
Gabija protein GajB


Mass: 57139.992 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus cereus VD045 (bacteria) / Gene: gajB / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: J8HQ06
#3: Protein
Gabija Anti-Defense 1


Mass: 34919.184 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus phage phi3T (virus) / Gene: phi3T_128 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A1P8CWZ3

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: 3D ARRAY / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Phage Phi3T Gad1 bound to the Bacillus cereus Gabija GajAB complexCOMPLEXall0RECOMBINANT
2Gabija GajAB complexCOMPLEX#1-#21RECOMBINANT
3Gad1 OctamerCOMPLEX#31RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.775 MDaNO
21.5 MDaNO
310.28 MDaNO
42
53
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Bacillus phage phi3T (virus)10736
52Bacillus cereus VD045 (bacteria)1053225
63Bacillus phage phi3T (virus)10736
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli BL21(DE3) (bacteria)469008
52Escherichia coli BL21(DE3) (bacteria)469008
63Escherichia coli BL21(DE3) (bacteria)469008
Buffer solutionpH: 7.5
Details: 20 mM HEPES-KOH pH 7.5, 20 mM KCl, and 1 mM TCEP-KOH
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1900 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 41.1 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 2.57 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 351193 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 170.99 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003539544
ELECTRON MICROSCOPYf_angle_d0.666153104
ELECTRON MICROSCOPYf_chiral_restr0.04265848
ELECTRON MICROSCOPYf_plane_restr0.00456764
ELECTRON MICROSCOPYf_dihedral_angle_d4.12535132

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