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- EMDB-74008: Visualization of PriA/PriB/DnaT complexes reveals mechanisms gove... -

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Basic information

Entry
Database: EMDB / ID: EMD-74008
TitleVisualization of PriA/PriB/DnaT complexes reveals mechanisms governing structure-specific assembly of the DNA replication restart primosome
Map data
Sample
  • Complex: Complex of PriA, PriB, DnaT, and replication fork
    • Protein or peptide: Replication restart protein PriB
    • Protein or peptide: Replication restart protein DnaT
    • Protein or peptide: Primosomal protein N'
    • DNA: DNA (33-MER)
    • DNA: DNA (33-MER)
    • DNA: DNA (11-MER)
  • Ligand: ZINC ION
KeywordsDNA replication / helicase / oligomer / DNA repair / REPLICATION
Function / homology
Function and homology information


pre-primosome complex / DnaB-DnaC-DnaT-PriA-PriC complex / DnaB-DnaC-DnaT-PriA-PriB complex / primosome complex / plasmid maintenance / DNA replication, synthesis of primer / protein homotrimerization / 3'-5' DNA helicase activity / DNA 3'-5' helicase / replication fork processing ...pre-primosome complex / DnaB-DnaC-DnaT-PriA-PriC complex / DnaB-DnaC-DnaT-PriA-PriB complex / primosome complex / plasmid maintenance / DNA replication, synthesis of primer / protein homotrimerization / 3'-5' DNA helicase activity / DNA 3'-5' helicase / replication fork processing / DNA replication initiation / helicase activity / response to gamma radiation / response to radiation / DNA-templated DNA replication / double-strand break repair / single-stranded DNA binding / DNA recombination / DNA replication / response to antibiotic / magnesium ion binding / ATP hydrolysis activity / DNA binding / RNA binding / zinc ion binding / ATP binding / identical protein binding
Similarity search - Function
Primosomal protein 1 / DnaT, DNA-binding domain / DnaT DNA-binding domain / Primosomal protein N' / PriA DNA helicase, Cys-rich region (CRR) domain / Primosomal protein N', 3' DNA-binding domain / Primosomal protein N, C-terminal domain / Primosomal protein N', 3' DNA-binding domain superfamily / : / 3'DNA-binding domain (3'BD) ...Primosomal protein 1 / DnaT, DNA-binding domain / DnaT DNA-binding domain / Primosomal protein N' / PriA DNA helicase, Cys-rich region (CRR) domain / Primosomal protein N', 3' DNA-binding domain / Primosomal protein N, C-terminal domain / Primosomal protein N', 3' DNA-binding domain superfamily / : / 3'DNA-binding domain (3'BD) / Primosomal protein N C-terminal domain / PriA DNA helicase Cys-rich region (CRR) domain / Primosomal protein N'-like, winged helix / Primosomal replication protein PriB / Another single-strand binding protein family / Single-strand binding (SSB) domain profile. / Primosome PriB/single-strand DNA-binding / DEAD/DEAH box helicase domain / DEAD/DEAH box helicase / Helicase conserved C-terminal domain / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / Nucleic acid-binding, OB-fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Replication restart protein PriB / Replication restart protein DnaT / Replication restart protein PriA
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.22 Å
AuthorsDuckworth AT / Keck JL / Grant T
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)RO1GM098885 United States
CitationJournal: Nat Commun / Year: 2026
Title: Visualization of the complete preprimosome reveals the structural mechanisms governing DNA replication restart.
Authors: Peter L Ducos / Alexander T Duckworth / Kenneth A Satyshur / James L Keck / Timothy Grant /
Abstract: Replication restart pathways reinitiate DNA replication processes following their premature termination. In Escherichia coli, this essential process begins with regulated assembly of the preprimosome ...Replication restart pathways reinitiate DNA replication processes following their premature termination. In Escherichia coli, this essential process begins with regulated assembly of the preprimosome complex, comprising the PriA, PriB, and DnaT proteins, onto an abandoned replication fork. Here, we present two distinct preprimosome structures. One represents an intermediate stage in preprimosome assembly with a single DnaT C-terminal domain (DnaT) bound to PriA/PriB/DNA. The second captures the mature preprimosome, in which filamentation of multiple DnaT molecules catalyzes the handoff of the single-stranded lagging-strand DNA from PriB to DnaT. The DnaT N-terminal domain forms a separate, independent oligomer in the mature structure. Taken together, our results detail the molecular mechanisms underlying replication restart initiation and regulation and suggest mechanistic similarities between DnaT and the canonical initiator protein DnaA.
History
DepositionNov 21, 2025-
Header (metadata) releaseMay 27, 2026-
Map releaseMay 27, 2026-
UpdateMay 27, 2026-
Current statusMay 27, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_74008.png

Downloads & links

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Map

FileDownload / File: emd_74008.map.gz / Format: CCP4 / Size: 129.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.09 Å/pix.
x 324 pix.
= 351.54 Å		1.09 Å/pix.
x 324 pix.
= 351.54 Å		1.09 Å/pix.
x 324 pix.
= 351.54 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.085 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.5
Minimum - Maximum-19.486350000000002 - 38.340240000000001
Average (Standard dev.)-0.000000000001315 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions324324324
Spacing324324324
CellA=B=C: 351.54 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_74008_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_74008_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 : Complex of PriA, PriB, DnaT, and replication fork

EntireName: Complex of PriA, PriB, DnaT, and replication fork
Components
  • Complex: Complex of PriA, PriB, DnaT, and replication fork
    • Protein or peptide: Replication restart protein PriB
    • Protein or peptide: Replication restart protein DnaT
    • Protein or peptide: Primosomal protein N'
    • DNA: DNA (33-MER)
    • DNA: DNA (33-MER)
    • DNA: DNA (11-MER)
  • Ligand: ZINC ION

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Supramolecule #1: Complex of PriA, PriB, DnaT, and replication fork

SupramoleculeName: Complex of PriA, PriB, DnaT, and replication fork / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #2, #1, #4, #3, #5-#6
Source (natural)Organism: Escherichia coli K-12 (bacteria)

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Macromolecule #1: Replication restart protein DnaT

MacromoleculeName: Replication restart protein DnaT / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 19.474025 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MSSRVLTPDV VGIDALVHDH QTVLAKAEGG VVAVFANNAP AFYAVTPARL AELLALEEKL ARPGSDVALD DQLYQEPQAA PVAVPMGKF AMYPDWQPDA DFIRLAALWG VALREPVTTE ELASFIAYWQ AEGKVFHHVQ WQQKLARSLQ IGRASNGGLP K RDVNTVSE PDSQIPPGFR G

UniProtKB: Replication restart protein DnaT

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Macromolecule #2: Replication restart protein PriB

MacromoleculeName: Replication restart protein PriB / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 11.459194 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MTNRLVLSGT VCRAPLRKVS PSGIPHCQFV LEHRSVQEEA GFHRQAWCQM PVIVSGHENQ AITHSITVGS RITVQGFISC HKAKNGLSK MVLHAEQIEL IDSGD

UniProtKB: Replication restart protein PriB

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Macromolecule #4: Primosomal protein N'

MacromoleculeName: Primosomal protein N' / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
EC number: Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 81.76582 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MPVAHVALPV PLPRTFDYLL PEGMTVKAGC RVRVPFGKQQ ERIGIVVSVS DASELPLNEL KAVVEVLDSE PVFTHSVWRL LLWAADYYH HPIGDVLFHA LPILLRQGRP AANAPMWYWF ATEQGQAVDL NSLKRSPKQQ QALAALRQGK IWRDQVATLE F NDAALQAL ...String:
MPVAHVALPV PLPRTFDYLL PEGMTVKAGC RVRVPFGKQQ ERIGIVVSVS DASELPLNEL KAVVEVLDSE PVFTHSVWRL LLWAADYYH HPIGDVLFHA LPILLRQGRP AANAPMWYWF ATEQGQAVDL NSLKRSPKQQ QALAALRQGK IWRDQVATLE F NDAALQAL RKKGLCDLAS ETPEFSDWRT NYAVSGERLR LNTEQATAVG AIHSAADTFS AWLLAGVTGS GKTEVYLSVL EN VLAQGKQ ALVMVPEIGL TPQTIARFRE RFNAPVEVLH SGLNDSERLS AWLKAKNGEA AIVIGTRSAL FTPFKNLGVI VID EEHDSS YKQQEGWRYH ARDLAVYRAH SEQIPIILGS ATPALETLCN VQQKKYRLLR LTRRAGNARP AIQHVLDLKG QKVQ AGLAP ALITRMRQHL QADNQVILFL NRRGFAPALL CHDCGWIAEC PRCDHYYTLH QAQHHLRCHH CDSQRPVPRQ CPSCG STHL VPVGLGTEQL EQTLAPLFPG VPISRIDRDT TSRKGALEQQ LAEVHRGGAR ILIGTQMLAK GHHFPDVTLV ALLDVD GAL FSADFRSAER FAQLYTQVAG RAGRAGKQGE VVLQTHHPEH PLLQTLLYKG YDAFAEQALA ERRMMQLPPW TSHVIVR AE DHNNQHAPLF LQQLRNLILS SPLADEKLWV LGPVPALAPK RGGRWRWQIL LQHPSRVRLQ HIINGTLALI NTIPDSRK V KWVLDVDPIE G

UniProtKB: Replication restart protein PriA

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Macromolecule #3: DNA (33-MER)

MacromoleculeName: DNA (33-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 12.206812 KDa
SequenceString:
(DG)(DC)(DC)(DG)(DC)(DA)(DG)(DA)(DC)(DT) (DC)(DA)(DT)(DT)(DT)(DA)(DG)(DC)(DC)(DC) (DT)(DT)(DA)(DT)(DC)(DC)(DG)(DT)(DA) (DT)(DT)(DG)(DC)(DG)(DG)(DT)(DC)(DT)(DC) (DG)

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Macromolecule #5: DNA (33-MER)

MacromoleculeName: DNA (33-MER) / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 12.399983 KDa
SequenceString:
(DC)(DG)(DA)(DG)(DA)(DC)(DC)(DG)(DC)(DA) (DA)(DT)(DA)(DC)(DG)(DG)(DA)(DT)(DA)(DA) (DG)(DG)(DG)(DC)(DT)(DG)(DA)(DG)(DC) (DA)(DC)(DG)(DC)(DC)(DG)(DA)(DC)(DG)(DA) (DA)

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Macromolecule #6: DNA (11-MER)

MacromoleculeName: DNA (11-MER) / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 4.567944 KDa
SequenceString:
(DT)(DT)(DC)(DG)(DT)(DC)(DG)(DG)(DC)(DG) (DT)(DG)(DC)(DT)(DC)

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Macromolecule #7: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 7 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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

Concentration1 mg/mL
BufferpH: 8
Details: 50 mM Tris-HCl, pH 8, 2 mM dithiothreitol, 5 mM ethylenediaminetetraacetic acid, 75 mM NaCl
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recording#0 - Image recording ID: 1 / #0 - Film or detector model: GATAN K3 (6k x 4k) / #0 - Number grids imaged: 1 / #0 - Number real images: 1988 / #0 - Average electron dose: 100.0 e/Å2 / #1 - Image recording ID: 2 / #1 - Film or detector model: GATAN K3 (6k x 4k) / #1 - Number grids imaged: 1 / #1 - Number real images: 1999 / #1 - Average electron dose: 100.0 e/Å2 / #1 - Details: 20 degree tilt
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Image recording ID1
Particle selectionNumber selected: 2200000
CTF correctionSoftware - Name: cisTEM / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER / Details: cisTEM ab initio
Final reconstructionNumber classes used: 15 / Resolution.type: BY AUTHOR / Resolution: 3.22 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM / Number images used: 857000
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: cisTEM
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cisTEM
FSC plot (resolution estimation)

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