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- PDB-8x51: Cryo-EM structure of Gabija GajA in complex with DNA(focused refi... -

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Basic information

Entry
Database: PDB / ID: 8x51
TitleCryo-EM structure of Gabija GajA in complex with DNA(focused refinement)
Components
  • DNA (5'-D(*AP*AP*AP*AP*AP*TP*AP*AP*CP*CP*GP*GP*GP*TP*TP*AP*TP*TP*AP*AP*A)-3')
  • DNA (5'-D(*TP*TP*TP*AP*AP*TP*AP*AP*CP*CP*CP*GP*GP*TP*TP*AP*TP*TP*TP*TP*T)-3')
  • Endonuclease GajA
KeywordsDNA BINDING PROTEIN/DNA / endonuclease / Complex / DNA BINDING / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / 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 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA / DNA (> 10) / Endonuclease GajA
Similarity search - Component
Biological speciesBacillus cereus VD045 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.92 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 16, 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

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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
E: DNA (5'-D(*TP*TP*TP*AP*AP*TP*AP*AP*CP*CP*CP*GP*GP*TP*TP*AP*TP*TP*TP*TP*T)-3')
F: DNA (5'-D(*AP*AP*AP*AP*AP*TP*AP*AP*CP*CP*GP*GP*GP*TP*TP*AP*TP*TP*AP*AP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)147,1206
Polymers147,0394
Non-polymers802
Water0
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.: 2
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: DNA chain DNA (5'-D(*TP*TP*TP*AP*AP*TP*AP*AP*CP*CP*CP*GP*GP*TP*TP*AP*TP*TP*TP*TP*T)-3')


Mass: 6393.154 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (5'-D(*AP*AP*AP*AP*AP*TP*AP*AP*CP*CP*GP*GP*GP*TP*TP*AP*TP*TP*AP*AP*A)-3')


Mass: 6487.263 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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 GajA with dsDNA / Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
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 FIELDBright-field microscopy / Nominal defocus max: 1800 nm / Nominal defocus min: 1400 nm
Image recordingElectron dose: 1.25 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.92 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 570317 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0078323
ELECTRON MICROSCOPYf_angle_d1.24311364
ELECTRON MICROSCOPYf_dihedral_angle_d21.0041362
ELECTRON MICROSCOPYf_chiral_restr0.071271
ELECTRON MICROSCOPYf_plane_restr0.0061296

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