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- PDB-8ojj: Cryo-EM structure of the DnaD-NTD tetramer -

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Basic information

Entry
Database: PDB / ID: 8ojj
TitleCryo-EM structure of the DnaD-NTD tetramer
ComponentsDNA replication protein DnaD
KeywordsDNA BINDING PROTEIN / Replication helicase loading / small cryo-EM structure
Function / homologyDnaD domain / DnaD-like domain superfamily / Replication initiation and membrane attachment / primosome complex / DNA replication, synthesis of primer / Winged helix-like DNA-binding domain superfamily / DNA replication protein DnaD
Function and homology information
Biological speciesBacillus subtilis subsp. subtilis str. 168 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.47 Å
AuthorsWinterhalter, C. / Pelliciari, S. / Cronin, N. / Costa, T.R.D. / Murray, H. / Ilangovan, A.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust204985/Z/16/Z United Kingdom
CitationJournal: Nucleic Acids Res / Year: 2023
Title: The DNA replication initiation protein DnaD recognises a specific strand of the Bacillus subtilis chromosome origin.
Authors: Charles Winterhalter / Simone Pelliciari / Daniel Stevens / Stepan Fenyk / Elie Marchand / Nora B Cronin / Panos Soultanas / Tiago R D Costa / Aravindan Ilangovan / Heath Murray /
Abstract: Genome replication is a fundamental biological activity shared by all organisms. Chromosomal replication proceeds bidirectionally from origins, requiring the loading of two helicases, one for each ...Genome replication is a fundamental biological activity shared by all organisms. Chromosomal replication proceeds bidirectionally from origins, requiring the loading of two helicases, one for each replisome. However, the molecular mechanisms underpinning helicase loading at bacterial chromosome origins (oriC) are unclear. Here we investigated the essential DNA replication initiation protein DnaD in the model organism Bacillus subtilis. A set of DnaD residues required for ssDNA binding was identified, and photo-crosslinking revealed that this ssDNA binding region interacts preferentially with one strand of oriC. Biochemical and genetic data support the model that DnaD recognizes a new single-stranded DNA (ssDNA) motif located in oriC, the DnaD Recognition Element (DRE). Considered with single particle cryo-electron microscopy (cryo-EM) imaging of DnaD, we propose that the location of the DRE within oriC orchestrates strand-specific recruitment of helicase during DNA replication initiation. These findings significantly advance our mechanistic understanding of bidirectional replication from a bacterial chromosome origin.
History
DepositionMar 24, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 17, 2023Provider: repository / Type: Initial release
Revision 1.1May 31, 2023Group: 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: DNA replication protein DnaD
B: DNA replication protein DnaD
C: DNA replication protein DnaD
D: DNA replication protein DnaD


Theoretical massNumber of molelcules
Total (without water)110,7034
Polymers110,7034
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area10630 Å2
ΔGint-71 kcal/mol
Surface area21370 Å2

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Components

#1: Protein
DNA replication protein DnaD


Mass: 27675.715 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis subsp. subtilis str. 168 (bacteria)
Strain: 168 / Gene: dnaD, BSU22350 / Production host: Escherichia coli (E. coli) / References: UniProt: P39787

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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: Homo Tetrameric complex of DnaD N-terminal domain / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Bacillus subtilis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7
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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2700 nm / Nominal defocus min: 700 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: EPU / Category: image acquisition
CTF correctionType: NONE
3D reconstructionResolution: 5.47 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 443529 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT

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