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- EMDB-76388: EcPriA bound to DNA replication fork with ssDNA lagging strand (C... -

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Basic information

Entry
Database: EMDB / ID: EMD-76388
TitleEcPriA bound to DNA replication fork with ssDNA lagging strand (CRR up)
Map data
Sample
  • Complex: EcPriA bound to DNA replication fork with ssDNA lagging strand (CRR up)
    • Protein or peptide: EcPriA
    • DNA: Leading template strand
    • DNA: Lagging template strand
    • DNA: Leading nascent strand
KeywordsReplication Fork / Helicase / DNA BINDING PROTEIN
Function / homology
Function and homology information


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 / DNA replication initiation / 3'-5' DNA helicase activity / response to gamma radiation / helicase activity ...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 / DNA replication initiation / 3'-5' DNA helicase activity / response to gamma radiation / helicase activity / replication fork processing / DNA-templated DNA replication / double-strand break repair / DNA recombination / DNA replication / response to antibiotic / ATP hydrolysis activity / DNA binding / zinc ion binding / ATP 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'-like, winged helix ...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 / 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 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Replication restart protein PriA
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsDuckworth AT / Deorio HR / Grant T / Keck JL
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM098885 United States
CitationJournal: Nucleic Acids Res / Year: 2026
Title: Domain dynamics within the PriA DNA helicase regulate DNA replication restart.
Authors: Haley R Deorio / Alexander T Duckworth / Gavin R Forsythe / Andrew Y Sung / Steve J Sandler / Timothy Grant / James L Keck /
Abstract: Prematurely terminated DNA replication processes in bacteria must be restarted for successful genome duplication. In Escherichia coli, the PriA DNA helicase orchestrates replication restart by ...Prematurely terminated DNA replication processes in bacteria must be restarted for successful genome duplication. In Escherichia coli, the PriA DNA helicase orchestrates replication restart by assembling the PriA/PriB/DnaT preprimosome complex onto abandoned DNA replication forks and reloading the replicative machinery. We show that the structure of the replication fork lagging strand-whether single- or double-stranded-influences the position of the cysteine-rich region in bound PriA (PriACRR). When PriA binds to synthetic replication forks with a single-stranded lagging strand, the PriACRR is found either in a state similar to free PriA or in a rotated position that encircles the lagging strand and allows PriB recruitment. Binding to a synthetic replication fork with a duplex lagging strand neither alters the PriACRR position nor promotes PriB binding, suggesting PriA must unwind duplex lagging-strand DNA to trigger PriACRR movement and subsequent preprimosome formation. PriA variants designed to destabilize the two PriACRR positions differentially affect ATPase activity, helicase function, PriB binding in vitro, and PriA activity in vivo. These results support a regulatory switch model in which the lagging-strand DNA structure modulates the PriACRR position, thereby governing PriA's biochemical and cellular activities.
History
DepositionMar 31, 2026-
Header (metadata) releaseOct 7, 2026-
Map releaseOct 7, 2026-
UpdateOct 7, 2026-
Current statusOct 7, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_76388.png

Downloads & links

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Map

FileDownload / File: emd_76388.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 288 pix.
= 239.904 Å		0.83 Å/pix.
x 288 pix.
= 239.904 Å		0.83 Å/pix.
x 288 pix.
= 239.904 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.833 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.25
Minimum - Maximum-3.2192273 - 7.533784
Average (Standard dev.)-0.0031840017 (±0.21981566)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 239.904 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_76388_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_76388_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 : EcPriA bound to DNA replication fork with ssDNA lagging strand (C...

EntireName: EcPriA bound to DNA replication fork with ssDNA lagging strand (CRR up)
Components
  • Complex: EcPriA bound to DNA replication fork with ssDNA lagging strand (CRR up)
    • Protein or peptide: EcPriA
    • DNA: Leading template strand
    • DNA: Lagging template strand
    • DNA: Leading nascent strand

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Supramolecule #1: EcPriA bound to DNA replication fork with ssDNA lagging strand (C...

SupramoleculeName: EcPriA bound to DNA replication fork with ssDNA lagging strand (CRR up)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 120 KDa

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Macromolecule #1: EcPriA

MacromoleculeName: EcPriA / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: DNA 3'-5' helicase
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MPVAHVALPV PLPRTFDYLL PEGMTVKAGC RVRVPFGKQQ ERIGIVVSVS DASELPLNEL KAVVEVLDSE PVFTHSVWRL LLWAADYYHH PIGDVLFHAL PILLRQGRPA ANAPMWYWFA TEQGQAVDLN SLKRSPKQQQ ALAALRQGKI WRDQVATLEF NDAALQALRK ...String:
MPVAHVALPV PLPRTFDYLL PEGMTVKAGC RVRVPFGKQQ ERIGIVVSVS DASELPLNEL KAVVEVLDSE PVFTHSVWRL LLWAADYYHH PIGDVLFHAL PILLRQGRPA ANAPMWYWFA TEQGQAVDLN SLKRSPKQQQ ALAALRQGKI WRDQVATLEF NDAALQALRK KGLCDLASET PEFSDWRTNY AVSGERLRLN TEQATAVGAI HSAADTFSAW LLAGVTGSGK TEVYLSVLEN VLAQGKQALV MVPEIGLTPQ TIARFRERFN APVEVLHSGL NDSERLSAWL KAKNGEAAIV IGTRSALFTP FKNLGVIVID EEHDSSYKQQ EGWRYHARDL AVYRAHSEQI PIILGSATPA LETLCNVQQK KYRLLRLTRR AGNARPAIQH VLDLKGQKVQ AGLAPALITR MRQHLQADNQ VILFLNRRGF APALLCHDCG WIAECPRCDH YYTLHQAQHH LRCHHCDSQR PVPRQCPSCG STHLVPVGLG TEQLEQTLAP LFPGVPISRI DRDTTSRKGA LEQQLAEVHR GGARILIGTQ MLAKGHHFPD VTLVALLDVD GALFSADFRS AERFAQLYTQ VAGRAGRAGK QGEVVLQTHH PEHPLLQTLL YKGYDAFAEQ ALAERRMMQL PPWTSHVIVR AEDHNNQHAP LFLQQLRNLI LSSPLADEKL WVLGPVPALA PKRGGRWRWQ ILLQHPSRVR LQHIINGTLA LINTIPDSRK VKWVLDVDPI EG

UniProtKB: Replication restart protein PriA

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Macromolecule #2: Leading template strand

MacromoleculeName: Leading template strand / type: dna / ID: 2 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
SequenceString:
CGAGACCGCA ATACGGATAA GGGCTGAGCA CGCCGACGAA

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Macromolecule #3: Lagging template strand

MacromoleculeName: Lagging template strand / type: dna / ID: 3 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
SequenceString:
GCCGCAGACT CATTTAGCCC TTATCCGTAT TGCGGTCTCG

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Macromolecule #4: Leading nascent strand

MacromoleculeName: Leading nascent strand / type: dna / ID: 4 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
SequenceString:
TTCGTCGGCG TGCTC

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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
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 65.0 e/Å2
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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: cisTEM / Type: NONE
Startup modelType of model: OTHER / Details: Ab initio
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM / Number images used: 90700
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: cisTEM
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cisTEM
Final 3D classificationSoftware - Name: cisTEM
FSC plot (resolution estimation)

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