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- PDB-8x5i: Structure of ATP/Mg2+ bound Gabija GajA -

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Basic information

Entry
Database: PDB / ID: 8x5i
TitleStructure of ATP/Mg2+ bound Gabija GajA
ComponentsEndonuclease GajA
KeywordsDNA BINDING PROTEIN / endonuclease
Function / homology
Function and homology information


endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / metal ion binding
Similarity search - Function
AAA domain, group 15 / : / Overcoming lysogenization defect protein-like, TOPRIM domain / OLD protein-like, TOPRIM domain / AAA ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / Endonuclease GajA
Similarity search - Component
Biological speciesBacillus cereus VD045 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.01 Å
AuthorsLi, J. / Wang, Z. / Wang, L.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Nature / Year: 2024
Title: Structures and activation mechanism of the Gabija anti-phage system.
Authors: Jing Li / Rui Cheng / Zhiming Wang / Wuliu Yuan / Jun Xiao / Xinyuan Zhao / Xinran Du / Shiyu Xia / Lianrong Wang / Bin Zhu / Longfei Wang /
Abstract: Prokaryotes have evolved intricate innate immune systems against phage infection. Gabija is a highly widespread prokaryotic defence system that consists of two components, GajA and GajB. GajA ...Prokaryotes have evolved intricate innate immune systems against phage infection. Gabija is a highly widespread prokaryotic defence system that consists of two components, GajA and GajB. GajA functions as a DNA endonuclease that is inactive in the presence of ATP. Here, to explore how the Gabija system is activated for anti-phage defence, we report its cryo-electron microscopy structures in five states, including apo GajA, GajA in complex with DNA, GajA bound by ATP, apo GajA-GajB, and GajA-GajB in complex with ATP and Mg. GajA is a rhombus-shaped tetramer with its ATPase domain clustered at the centre and the topoisomerase-primase (Toprim) domain located peripherally. ATP binding at the ATPase domain stabilizes the insertion region within the ATPase domain, keeping the Toprim domain in a closed state. Upon ATP depletion by phages, the Toprim domain opens to bind and cleave the DNA substrate. GajB, which docks on GajA, is activated by the cleaved DNA, ultimately leading to prokaryotic cell death. Our study presents a mechanistic landscape of Gabija activation.
History
DepositionNov 17, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 28, 2024Provider: repository / Type: Initial release
Revision 1.1Apr 3, 2024Group: 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
Revision 1.2May 22, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Oct 16, 2024Group: Data collection / Structure summary / Category: em_admin / pdbx_entry_details / struct
Item: _em_admin.last_update / _em_admin.title ..._em_admin.last_update / _em_admin.title / _pdbx_entry_details.has_protein_modification / _struct.title

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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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)270,44412
Polymers268,3184
Non-polymers2,1268
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Endonuclease GajA / Gabija protein GajA / Nicking endonuclease GajA


Mass: 67079.469 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus cereus VD045 (bacteria) / Gene: gajA, IIE_04982 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: J8H9C1, Hydrolases; Acting on ester bonds
#2: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: Binary complex of Gabija with ATP / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Bacillus cereus VD045 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 1600 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.21rc1_5127 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.01 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 4388286 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 75.02 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.008717426
ELECTRON MICROSCOPYf_angle_d1.210923447
ELECTRON MICROSCOPYf_chiral_restr0.06952567
ELECTRON MICROSCOPYf_plane_restr0.00782944
ELECTRON MICROSCOPYf_dihedral_angle_d7.81692258

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