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- PDB-8fak: DNA replication fork binding triggers structural changes in the P... -

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Basic information

Entry
Database: PDB / ID: 8fak
TitleDNA replication fork binding triggers structural changes in the PriA DNA helicase that regulate the PriA-PriB replication restart pathway in E. coli
Components
  • DNA (5'-D(P*CP*AP*GP*AP*CP*TP*CP*AP*TP*TP*TP*AP*GP*CP*CP*CP*TP*TP*AP*TP*CP*CP*G)-3')
  • DNA (5'-D(P*CP*GP*GP*AP*TP*AP*AP*GP*GP*GP*CP*TP*GP*AP*GP*CP*AP*CP*GP*CP*CP*GP*A)-3')
  • DNA (5'-D(P*TP*CP*GP*GP*CP*GP*TP*GP*CP*TP*C)-3')
  • Primosomal protein N'
  • Primosomal replication protein N
KeywordsHELICASE/DNA / PriA / PriB / replication restart / E. coli / HELICASE-DNA complex
Function / homology
Function and homology information


pre-primosome complex / DnaB-DnaC-DnaT-PriA-PriC complex / DnaB-DnaC-DnaT-PriA-PriB complex / plasmid maintenance / primosome complex / DNA replication, synthesis of primer / DNA 3'-5' helicase / 3'-5' DNA helicase activity / replication fork processing / DNA replication initiation ...pre-primosome complex / DnaB-DnaC-DnaT-PriA-PriC complex / DnaB-DnaC-DnaT-PriA-PriB complex / plasmid maintenance / primosome complex / DNA replication, synthesis of primer / DNA 3'-5' helicase / 3'-5' DNA helicase activity / replication fork processing / DNA replication initiation / response to gamma radiation / response to radiation / helicase activity / DNA-templated DNA replication / double-strand break repair / single-stranded DNA binding / DNA recombination / DNA replication / response to antibiotic / ATP hydrolysis activity / DNA binding / zinc ion binding / ATP binding / identical protein binding
Similarity search - Function
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' / 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 PriA
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.22 Å
AuthorsDuckworth, A.T. / Ducos, P.L. / McMillan, S.D. / Satyshur, K.A. / Blumenthal, K.H. / Deorio, H.R. / Larson, J.A. / Sandler, S.J. / Grant, T. / Keck, J.L.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM098885 United States
CitationJournal: Nat Commun / Year: 2023
Title: Replication fork binding triggers structural changes in the PriA helicase that govern DNA replication restart in E. coli.
Authors: Alexander T Duckworth / Peter L Ducos / Sarah D McMillan / Kenneth A Satyshur / Katelien H Blumenthal / Haley R Deorio / Joseph A Larson / Steven J Sandler / Timothy Grant / James L Keck /
Abstract: Bacterial replisomes often dissociate from replication forks before chromosomal replication is complete. To avoid the lethal consequences of such situations, bacteria have evolved replication restart ...Bacterial replisomes often dissociate from replication forks before chromosomal replication is complete. To avoid the lethal consequences of such situations, bacteria have evolved replication restart pathways that reload replisomes onto prematurely terminated replication forks. To understand how the primary replication restart pathway in E. coli (PriA-PriB) selectively acts on replication forks, we determined the cryogenic-electron microscopy structure of a PriA/PriB/replication fork complex. Replication fork specificity arises from extensive PriA interactions with each arm of the branched DNA. These interactions reshape the PriA protein to create a pore encircling single-stranded lagging-strand DNA while also exposing a surface of PriA onto which PriB docks. Together with supporting biochemical and genetic studies, the structure reveals a switch-like mechanism for replication restart initiation in which restructuring of PriA directly couples replication fork recognition to PriA/PriB complex formation to ensure robust and high-fidelity replication re-initiation.
History
DepositionNov 28, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 10, 2023Provider: repository / Type: Initial release
Revision 1.1May 24, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Oct 23, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Primosomal replication protein N
B: Primosomal replication protein N
G: DNA (5'-D(P*CP*AP*GP*AP*CP*TP*CP*AP*TP*TP*TP*AP*GP*CP*CP*CP*TP*TP*AP*TP*CP*CP*G)-3')
H: Primosomal protein N'
J: DNA (5'-D(P*CP*GP*GP*AP*TP*AP*AP*GP*GP*GP*CP*TP*GP*AP*GP*CP*AP*CP*GP*CP*CP*GP*A)-3')
Z: DNA (5'-D(P*TP*CP*GP*GP*CP*GP*TP*GP*CP*TP*C)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)133,9908
Polymers133,8596
Non-polymers1312
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 3 molecules ABH

#1: Protein Primosomal replication protein N / PriB


Mass: 11459.194 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: priB, b4201, JW4159 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P07013
#3: 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 (strain K12) (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

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DNA chain , 3 types, 3 molecules GJZ

#2: DNA chain DNA (5'-D(P*CP*AP*GP*AP*CP*TP*CP*AP*TP*TP*TP*AP*GP*CP*CP*CP*TP*TP*AP*TP*CP*CP*G)-3')


Mass: 12206.812 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA (5'-D(P*CP*GP*GP*AP*TP*AP*AP*GP*GP*GP*CP*TP*GP*AP*GP*CP*AP*CP*GP*CP*CP*GP*A)-3')


Mass: 12399.983 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: DNA chain DNA (5'-D(P*TP*CP*GP*GP*CP*GP*TP*GP*CP*TP*C)-3')


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

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Non-polymers , 1 types, 2 molecules

#6: 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 modificationY

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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, and replication fork / Type: COMPLEX / Entity ID: #1-#5 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.134 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21 (DE3)
Buffer solutionpH: 8
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: Quantifoil
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: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recording
IDImaging-IDElectron dose (e/Å2)Film or detector modelNum. of grids imagedNum. of real imagesDetector mode
11100GATAN K3 (6k x 4k)11600
2160FEI FALCON III (4k x 4k)11200INTEGRATING

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Processing

SoftwareName: UCSF ChimeraX / Version: 1.4/v9 / Classification: model building / URL: https://www.rbvi.ucsf.edu/chimerax/ / Os: Linux / Type: package
EM software
IDNameVersionCategory
1cisTEM2.0.0particle selection
2SerialEMimage acquisition
4cisTEM2.0.0CTF correction
7UCSF Chimeramodel fitting
9cisTEM2.0.0initial Euler assignment
10cisTEM2.0.0final Euler assignment
12cisTEM2.0.03D reconstruction
13PHENIXmodel refinement
Image processingDetails: Processing was performed in the same way for K3 and Falcon 3 data.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1500000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.22 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 190000 / Details: Highest resolution used in refinement was 4.4A / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL

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