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- PDB-29km: Cryo-EM structure of Bacillus subtilis DnaB -

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Basic information

Entry
Database: PDB / ID: 29km
TitleCryo-EM structure of Bacillus subtilis DnaB
ComponentsReplicative helicase loading/DNA remodeling protein DnaB
KeywordsDNA BINDING PROTEIN / Apo DnaB structure / Bacillus subtilis DNA replication / Helicase loader
Function / homology
Function and homology information


primosome complex / DNA replication, synthesis of primer / DNA replication / DNA binding / plasma membrane / cytoplasm
Similarity search - Function
: / DnaB N-terminal winged helix domain / DnaD domain / DnaD-like domain superfamily / Replication initiation and membrane attachment
Similarity search - Domain/homology
Replicative helicase loading/DNA remodeling protein DnaB
Similarity search - Component
Biological speciesBacillus subtilis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsCampoy, R.R. / Guyet, A. / Pelliciari, S. / Murray, H. / Ilangovan, A.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust225811/Z/22/Z United Kingdom
CitationJournal: Nucleic Acids Res / Year: 2026
Title: Bacillus subtilis DnaB forms multiple protein-protein interactions essential for DNA replication initiation.
Authors: Aurélie Guyet / Reyes Ruiz Campoy / Petra Manja / Frederic D Schramm / Simone Pelliciari / Stepan Fenyk / Yuanyuan Li / Charles Winterhalter / Aravindan Ilangovan / Heath Murray /
Abstract: DNA replication is initiated at specific chromosomal loci termed origins. In bacteria, the master replication initiation protein DnaA unwinds the origin (oriC), allowing a pair of replicative ...DNA replication is initiated at specific chromosomal loci termed origins. In bacteria, the master replication initiation protein DnaA unwinds the origin (oriC), allowing a pair of replicative helicases to be loaded around each strand of the DNA duplex. The molecular mechanisms for managing bacterial helicase loading at oriC are unclear. Here we have investigated the role of the essential accessory helicase loader DnaB in Bacillus subtilis. By identifying and characterizing DnaB residues that are critical for its role during DNA replication initiation, we have located three necessary protein-protein interactions that DnaB makes with initiation proteins DnaA, DnaD, and DnaI. Combining single particle cryo-electron microscopy, AlphaFold3 predictions, and two-hybrid interaction analyses, the data suggests that DnaB acts as an interaction hub to orchestrate dual helicase loading at the origin. We propose a model for DNA replication initiation in B. subtilis and related Firmicutes pathogens that employ DnaB-type helicase loaders.
History
DepositionMar 18, 2026Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 15, 2026Provider: repository / Type: Initial release
Revision 1.0Jul 15, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jul 15, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jul 15, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jul 15, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jul 15, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jul 15, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Replicative helicase loading/DNA remodeling protein DnaB
B: Replicative helicase loading/DNA remodeling protein DnaB
C: Replicative helicase loading/DNA remodeling protein DnaB
D: Replicative helicase loading/DNA remodeling protein DnaB


Theoretical massNumber of molelcules
Total (without water)219,8434
Polymers219,8434
Non-polymers00
Water00
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
Replicative helicase loading/DNA remodeling protein DnaB / Replication initiation and membrane attachment protein / Replicative helicase loader DnaB


Mass: 54960.770 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: DnaB chains A and D / Source: (gene. exp.) Bacillus subtilis (bacteria) / Gene: dnaB, BSU28990 / Production host: Escherichia coli (E. coli) / References: UniProt: P07908
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: Bacillus subtilis DnaB / Type: ORGANELLE OR CELLULAR COMPONENT / Details: Apo / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.104 MDa / Experimental value: NO
Source (natural)Organism: Bacillus subtilis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Apo DnaB
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Details: Leica EM GP2

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 700 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 12516

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.7.1particle selection
2EPUimage acquisition
4cryoSPARC4.7.1CTF correction
7UCSF ChimeraX10model fitting
12cryoSPARC3D reconstruction
13PHENIX1.21.2_5419model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2578235
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 144168 / Num. of class averages: 3 / Symmetry type: POINT
Atomic model buildingB value: 139.5 / Protocol: RIGID BODY FIT / Space: REAL
Details: Initial rigid body fitting was done using Chimera X (Fit to Map). After retracing terminal residues, as well as rebuilding around Arg57, further modelling and real space refinement was used ...Details: Initial rigid body fitting was done using Chimera X (Fit to Map). After retracing terminal residues, as well as rebuilding around Arg57, further modelling and real space refinement was used to refine the final model.
Atomic model buildingChain residue range: 7-288 / Details: Truncated alphafold DnaB tetramer prediction / Source name: AlphaFold / Type: in silico model
RefinementHighest resolution: 3.1 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0027504
ELECTRON MICROSCOPYf_angle_d0.40410148
ELECTRON MICROSCOPYf_dihedral_angle_d11.3832874
ELECTRON MICROSCOPYf_chiral_restr0.0361102
ELECTRON MICROSCOPYf_plane_restr0.0031316

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