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- EMDB-16914: Cryo-EM structure of the DnaD-NTD tetramer -

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Basic information

Entry
Database: EMDB / ID: EMD-16914
TitleCryo-EM structure of the DnaD-NTD tetramer
Map data
Sample
  • Complex: Homo Tetrameric complex of DnaD N-terminal domain
    • Protein or peptide: DNA replication protein DnaD
KeywordsReplication helicase loading / small cryo-EM structure / DNA BINDING PROTEIN
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 (bacteria) / Bacillus subtilis subsp. subtilis str. 168 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.47 Å
AuthorsWinterhalter C / Pelliciari S / Cronin N / Costa TRD / Murray H / Ilangovan A
Funding support United Kingdom, 1 items
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, 2023-
Header (metadata) releaseMay 17, 2023-
Map releaseMay 17, 2023-
UpdateMay 31, 2023-
Current statusMay 31, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16914.png

Downloads & links

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Map

FileDownload / File: emd_16914.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.831 Å
Density
Contour LevelBy AUTHOR: 0.27
Minimum - Maximum-0.7487046 - 1.295883
Average (Standard dev.)0.0002561508 (±0.039995626)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 212.736 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_16914_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_16914_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Homo Tetrameric complex of DnaD N-terminal domain

EntireName: Homo Tetrameric complex of DnaD N-terminal domain
Components
  • Complex: Homo Tetrameric complex of DnaD N-terminal domain
    • Protein or peptide: DNA replication protein DnaD

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Supramolecule #1: Homo Tetrameric complex of DnaD N-terminal domain

SupramoleculeName: Homo Tetrameric complex of DnaD N-terminal domain / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Bacillus subtilis (bacteria)

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Macromolecule #1: DNA replication protein DnaD

MacromoleculeName: DNA replication protein DnaD / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Bacillus subtilis subsp. subtilis str. 168 (bacteria)
Strain: 168
Molecular weightTheoretical: 27.675715 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKKQQFIDMQ EQGTSTIPNL LLTHYKQLGL NETELILLLK IKMHLEKGSY FPTPNQLQEG MSISVEECTN RLRMFIQKGF LFIEECEDQ NGIKFEKYSL QPLWGKLYEY IQLAQNQTQE RKAEGEQKSL YTIFEEEFAR PLSPLECETL AIWQDQDQHD A QLIKHALK ...String:
MKKQQFIDMQ EQGTSTIPNL LLTHYKQLGL NETELILLLK IKMHLEKGSY FPTPNQLQEG MSISVEECTN RLRMFIQKGF LFIEECEDQ NGIKFEKYSL QPLWGKLYEY IQLAQNQTQE RKAEGEQKSL YTIFEEEFAR PLSPLECETL AIWQDQDQHD A QLIKHALK EAVLSGKLSF RYIDRILFEW KKNGLKTVEQ AKIHSQKFRR VQAKQNEPQK EYKRQVPFYN WLEQ

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.7 µm / Nominal defocus min: 0.7000000000000001 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 5.47 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 443529
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT
Output model

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

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