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- PDB-9zbt: Visualization of PriA/PriB/DnaT complexes reveals mechanisms gove... -

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Basic information

Entry
Database: PDB / ID: 9zbt
TitleVisualization of PriA/PriB/DnaT complexes reveals mechanisms governing structure-specific assembly of the DNA replication restart primosome
Components
  • (DNA (33-MER)) x 2
  • (Replication restart protein ...) x 2
  • DNA (11-MER)
  • Primosomal protein N'
KeywordsREPLICATION / DNA replication / helicase / oligomer / DNA repair
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
DNA / DNA (> 10) / Replication restart protein PriB / Replication restart protein DnaT / Replication restart protein PriA
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.22 Å
AuthorsDuckworth, A.T. / Keck, J.L. / Grant, T.
Funding support United States, 1items
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, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 27, 2026Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0May 27, 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

Downloads & links

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Assembly

Deposited unit
D: Replication restart protein DnaT
A: Replication restart protein PriB
B: Replication restart protein PriB
G: DNA (33-MER)
H: Primosomal protein N'
J: DNA (33-MER)
Z: DNA (11-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)153,4649
Polymers153,3337
Non-polymers1312
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Replication restart protein ... , 2 types, 3 molecules DAB

#1: Protein Replication restart protein DnaT / Primosomal protein DnaT / Primosomal protein i


Mass: 19474.025 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: dnaT, b4362, JW4326 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0A8J2
#2: Protein Replication restart protein PriB / Primosomal replication protein n


Mass: 11459.194 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: priB, b4201, JW4159 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P07013

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DNA chain , 3 types, 3 molecules GJZ

#3: DNA chain DNA (33-MER)


Mass: 12206.812 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: DNA chain DNA (33-MER)


Mass: 12399.983 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#6: DNA chain DNA (11-MER)


Mass: 4567.944 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Protein / Non-polymers , 2 types, 3 molecules H

#4: Protein Primosomal protein N' / ATP-dependent helicase PriA / Replication factor Y


Mass: 81765.820 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: priA, b3935, JW3906 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P17888, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
#7: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestN
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: Complex of PriA, PriB, DnaT, and replication fork / Type: COMPLEX / Entity ID: #2, #1, #4, #3, #5-#6 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
Details: 50 mM Tris-HCl, pH 8, 2 mM dithiothreitol, 5 mM ethylenediaminetetraacetic acid, 75 mM NaCl
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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: 2500 nm / Nominal defocus min: 500 nm
Specimen holderCryogen: NITROGEN
Image recording
IDImaging-IDElectron dose (e/Å2)Film or detector modelNum. of grids imagedNum. of real imagesDetails (eV)
11100GATAN K3 (6k x 4k)11988
21100GATAN K3 (6k x 4k)1199920 degree tilt
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameCategory
1cisTEMparticle selection
2SerialEMimage acquisition
4cisTEMCTF correction
7UCSF ChimeraXmodel fitting
8Cootmodel fitting
10cisTEMinitial Euler assignment
11cisTEMfinal Euler assignment
12cisTEMclassification
13cisTEM3D reconstruction
14PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2200000
3D reconstructionResolution: 3.22 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 857000 / Num. of class averages: 15 / Symmetry type: POINT
RefinementHighest resolution: 3.22 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0029973
ELECTRON MICROSCOPYf_angle_d0.46213874
ELECTRON MICROSCOPYf_dihedral_angle_d22.2171931
ELECTRON MICROSCOPYf_chiral_restr0.041555
ELECTRON MICROSCOPYf_plane_restr0.0031528

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