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- EMDB-28959: DNA replication fork binding triggers structural changes in the P... -

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Basic information

Entry
Database: EMDB / ID: EMD-28959
TitleDNA replication fork binding triggers structural changes in the PriA DNA helicase that regulate the PriA-PriB replication restart pathway in E. coli
Map datafinal sharpened map
Sample
  • Complex: Complex of PriA, PriB, and replication fork
    • Protein or peptide: Primosomal replication protein N
    • DNA: 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')
    • Protein or peptide: Primosomal protein N'
    • DNA: 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: DNA (5'-D(P*TP*CP*GP*GP*CP*GP*TP*GP*CP*TP*C)-3')
  • Ligand: ZINC ION
KeywordsPriA / 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 / 3'-5' DNA helicase activity / DNA unwinding involved in DNA replication / 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 / 3'-5' DNA helicase activity / DNA unwinding involved in DNA replication / replication fork processing / DNA replication initiation / response to gamma radiation / helicase activity / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / response to radiation / DNA-templated DNA replication / double-strand break repair / single-stranded DNA binding / DNA recombination / DNA replication / hydrolase activity / response to antibiotic / 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
Primosomal replication protein N / Primosomal protein N'
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / Escherichia coli (strain K12) (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.22 Å
AuthorsDuckworth AT / Ducos PL / McMillan SD / Satyshur KA / Blumenthal KH / Deorio HR / Larson JA / Sandler SJ / Grant T / Keck JL
Funding support United States, 1 items
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, 2022-
Header (metadata) releaseMay 10, 2023-
Map releaseMay 10, 2023-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28959.png

Downloads & links

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Map

FileDownload / File: emd_28959.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationfinal sharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.08 Å/pix.
x 192 pix.
= 207.168 Å		1.08 Å/pix.
x 192 pix.
= 207.168 Å		1.08 Å/pix.
x 192 pix.
= 207.168 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.079 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.5
Minimum - Maximum-6.5150013 - 12.985538999999999
Average (Standard dev.)0.003259282 (±0.4999796)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 207.168 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: unfiltered / unsharpened half map 1

Fileemd_28959_half_map_1.map
Annotationunfiltered / unsharpened half map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: unfiltered / unsharpened half map 2

Fileemd_28959_half_map_2.map
Annotationunfiltered / unsharpened half map 2
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, and replication fork

EntireName: Complex of PriA, PriB, and replication fork
Components
  • Complex: Complex of PriA, PriB, and replication fork
    • Protein or peptide: Primosomal replication protein N
    • DNA: 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')
    • Protein or peptide: Primosomal protein N'
    • DNA: 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: DNA (5'-D(P*TP*CP*GP*GP*CP*GP*TP*GP*CP*TP*C)-3')
  • Ligand: ZINC ION

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

SupramoleculeName: Complex of PriA, PriB, and replication fork / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12
Molecular weightTheoretical: 134 KDa

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Macromolecule #1: Primosomal replication protein N

MacromoleculeName: Primosomal replication protein N / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (strain K12) (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: Primosomal replication protein N

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Macromolecule #3: Primosomal protein N'

MacromoleculeName: Primosomal protein N' / type: protein_or_peptide / ID: 3 / 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 (strain K12) (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: Primosomal protein N'

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Macromolecule #2: DNA (5'-D(P*CP*AP*GP*AP*CP*TP*CP*AP*TP*TP*TP*AP*GP*CP*CP*CP*TP*TP...

MacromoleculeName: 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')
type: dna / ID: 2 / 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 #4: DNA (5'-D(P*CP*GP*GP*AP*TP*AP*AP*GP*GP*GP*CP*TP*GP*AP*GP*CP*AP*CP...

MacromoleculeName: 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')
type: dna / ID: 4 / 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 #5: DNA (5'-D(P*TP*CP*GP*GP*CP*GP*TP*GP*CP*TP*C)-3')

MacromoleculeName: DNA (5'-D(P*TP*CP*GP*GP*CP*GP*TP*GP*CP*TP*C)-3') / type: dna / ID: 5 / 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 #6: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 6 / 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
GridModel: Quantifoil / Material: GOLD / Support film - Material: GOLD / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
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: 1600 / #0 - Average electron dose: 100.0 e/Å2 / #1 - Image recording ID: 2 / #1 - Film or detector model: FEI FALCON III (4k x 4k) / #1 - Detector mode: INTEGRATING / #1 - Number grids imaged: 1 / #1 - Number real images: 1200 / #1 - Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Image recording ID1
DetailsProcessing was performed in the same way for K3 and Falcon 3 data.
Particle selectionNumber selected: 1500000
Startup modelType of model: NONE / Details: cisTEM ab-inito was used.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.22 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM (ver. 2.0.0) / Details: Highest resolution used in refinement was 4.4A / Number images used: 190000
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: cisTEM (ver. 2.0.0)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cisTEM (ver. 2.0.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8fak:
DNA replication fork binding triggers structural changes in the PriA DNA helicase that regulate the PriA-PriB replication restart pathway in E. coli

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