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- EMDB-43082: Ecoli DnaB helicase and Phage Lambda loader P with ADP-Mg in a 6:... -

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Entry
Database: EMDB / ID: EMD-43082
TitleEcoli DnaB helicase and Phage Lambda loader P with ADP-Mg in a 6:5 stoichiometry ratio
Map dataThe attached map has been sharpened using the DeepEMhancer software.
Sample
  • Complex: Ecoli DnaB helicase and Phage Lambda loader P with ADP-Mg in a 6:5 stoichiometry ratio.
    • Protein or peptide: Replicative DNA helicase
    • Protein or peptide: Helicase loader
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
KeywordsHexameric DnaB helicase / Phage Lambda P helicase loader / bacterial DNA replication initiation / auto inhibition / DNA BINDING PROTEIN
Function / homology
Function and homology information


DnaB-DnaC complex / DnaB-DnaC-Rep-PriC complex / DnaB-DnaG complex / DnaB-DnaC-DnaT-PriA-PriC complex / DnaB-DnaC-DnaT-PriA-PriB complex / DNA helicase complex / primosome complex / DNA 5'-3' helicase / bidirectional double-stranded viral DNA replication / DNA replication, synthesis of primer ...DnaB-DnaC complex / DnaB-DnaC-Rep-PriC complex / DnaB-DnaG complex / DnaB-DnaC-DnaT-PriA-PriC complex / DnaB-DnaC-DnaT-PriA-PriB complex / DNA helicase complex / primosome complex / DNA 5'-3' helicase / bidirectional double-stranded viral DNA replication / DNA replication, synthesis of primer / replisome / response to ionizing radiation / replication fork processing / DNA replication initiation / DNA helicase activity / helicase activity / 5'-3' DNA helicase activity / DNA replication / ATP hydrolysis activity / DNA binding / ATP binding / identical protein binding / cytosol
Similarity search - Function
Replication P / Replication protein P / DNA helicase, DnaB type / DNA helicase, DnaB-like, N-terminal / DnaB-like helicase N terminal domain / DNA helicase, DnaB-like, N-terminal domain superfamily / DNA helicase DnaB, N-terminal/DNA primase DnaG, C-terminal / DnaB-like helicase C terminal domain / DNA helicase, DnaB-like, C-terminal / Superfamily 4 helicase domain profile. ...Replication P / Replication protein P / DNA helicase, DnaB type / DNA helicase, DnaB-like, N-terminal / DnaB-like helicase N terminal domain / DNA helicase, DnaB-like, N-terminal domain superfamily / DNA helicase DnaB, N-terminal/DNA primase DnaG, C-terminal / DnaB-like helicase C terminal domain / DNA helicase, DnaB-like, C-terminal / Superfamily 4 helicase domain profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Helicase loader / Replicative DNA helicase DnaB
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / Escherichia phage Lambda (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.84 Å
AuthorsShatarupa A / Brown D / Olinares PDB / Chase J / Isiorho E / Chait BT / Jeruzalmi D
Funding support United States, 1 items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)1818255 United States
Citation
Journal: Nucleic Acids Res / Year: 2025
Title: Distinct quaternary states, intermediates, and autoinhibition during loading of the DnaB-replicative helicase by the phage λP helicase loader.
Authors: Abhipsa Shatarupa / Dhanjai Brown / Paul Dominic B Olinares / Jillian Chase / Eta Isiorho / Brian T Chait / David Jeruzalmi /
Abstract: Replicative helicases need loader proteins to assemble at DNA replication origins. Multiple copies of the bacteriophage λP (P) loader bind and load the Escherichia coli DnaB (B) replicative helicase ...Replicative helicases need loader proteins to assemble at DNA replication origins. Multiple copies of the bacteriophage λP (P) loader bind and load the Escherichia coli DnaB (B) replicative helicase onto single-stranded (ss) DNA from the replication origin. We find that the E. coli DnaB•λP complex exists in two forms: B6P5 and B6P6. In the 2.66 Å cryo-EM structure of B6P5, five λP loader copies form a crown-like shape that tightly grips DnaB. In this complex, the closed, planar DnaB is reconfigured into an open spiral with a large enough breach to allow ssDNA to enter an internal chamber. Transition to the open spiral involves λP-induced changes to the Docking Helix (DH)-Linker Helix (LH) interface. Unexpectedly, one λP chain in B6P5 is positioned across the breach. The disposition of this λP chain implies a complex pathway for entry of a replication-origin-derived ssDNA "bubble" ssDNA into the B6P5 complex. We propose that the B6P6 complex is an early intermediate in helicase activation in which neither DnaB nor λP has reached its final form. In this complex, DnaB adopts a partially open, ajar planar configuration. λP in B6P6 interacts more loosely with DnaB. The ssDNA- and ATP-binding sites in both complexes are not correctly configured for binding or hydrolysis. Our findings detail the distinct conformations of B6P6 and B6P5, allowing us to propose a structural model for the transition from an ajar planar to an open spiral configuration in the helicase loading pathway.
#1: Journal: Biorxiv / Year: 2025
Title: An Autoinhibited Conformation of the DnaB-Replicative Helicase- phage LP Helicase Loader Complex
Authors: Brown D / Shatarupa A / Olinares PDB / Chase J / Isiorho E / Chait BT / Jeruzalmi D
History
DepositionDec 9, 2023-
Header (metadata) releaseMar 4, 2026-
Map releaseMar 4, 2026-
UpdateMar 4, 2026-
Current statusMar 4, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43082.png

Downloads & links

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Map

FileDownload / File: emd_43082.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThe attached map has been sharpened using the DeepEMhancer software.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 320 pix.
= 346.56 Å		1.08 Å/pix.
x 320 pix.
= 346.56 Å		1.08 Å/pix.
x 320 pix.
= 346.56 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.083 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.135
Minimum - Maximum-0.0018494652 - 2.2886662
Average (Standard dev.)0.00223351 (±0.034839943)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 346.56 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_43082_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_43082_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : Ecoli DnaB helicase and Phage Lambda loader P with ADP-Mg in a 6:...

EntireName: Ecoli DnaB helicase and Phage Lambda loader P with ADP-Mg in a 6:5 stoichiometry ratio.
Components
  • Complex: Ecoli DnaB helicase and Phage Lambda loader P with ADP-Mg in a 6:5 stoichiometry ratio.
    • Protein or peptide: Replicative DNA helicase
    • Protein or peptide: Helicase loader
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Ecoli DnaB helicase and Phage Lambda loader P with ADP-Mg in a 6:...

SupramoleculeName: Ecoli DnaB helicase and Phage Lambda loader P with ADP-Mg in a 6:5 stoichiometry ratio.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Molecular weightTheoretical: 446.94 KDa

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Macromolecule #1: Replicative DNA helicase

MacromoleculeName: Replicative DNA helicase / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 52.450945 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MAGNKPFNKQ QAEPRERDPQ VAGLKVPPHS IEAEQSVLGG LMLDNERWDD VAERVVADDF YTRPHRHIFT EMARLQESGS PIDLITLAE SLERQGQLDS VGGFAYLAEL SKNTPSAANI SAYADIVRER AVVREMISVA NEIAEAGFDP QGRTSEDLLD L AESRVFKI ...String:
MAGNKPFNKQ QAEPRERDPQ VAGLKVPPHS IEAEQSVLGG LMLDNERWDD VAERVVADDF YTRPHRHIFT EMARLQESGS PIDLITLAE SLERQGQLDS VGGFAYLAEL SKNTPSAANI SAYADIVRER AVVREMISVA NEIAEAGFDP QGRTSEDLLD L AESRVFKI AESRANKDEG PKNIADVLDA TVARIEQLFQ QPHDGVTGVN TGYDDLNKKT AGLQPSDLII VAARPSMGKT TF AMNLVEN AAMLQDKPVL IFSLEMPSEQ IMMRSLASLS RVDQTKIRTG QLDDEDWARI SGTMGILLEK RNIYIDDSSG LTP TEVRSR ARRIAREHGG IGLIMIDYLQ LMRVPALSDN RTLEIAEISR SLKALAKELN VPVVALSQLN RSLEQRADKR PVNS DLRES GSIEQDADLI MFIYRDEVYH ENSDLKGIAE IIIGKQRNGP IGTVRLTFNG QWSRFDNYAG PQYDDE

UniProtKB: Replicative DNA helicase DnaB

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Macromolecule #2: Helicase loader

MacromoleculeName: Helicase loader / type: protein_or_peptide / ID: 2 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Escherichia phage Lambda (virus)
Molecular weightTheoretical: 26.551326 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MENIAAQMVN FDREQMRRIA NNMPEQYDEK PQVQQVAQII NGVFSQLLAT FPASLANRDQ NEVNEIRRQW VLAFRENGIT TMEQVNAGM RVARRQNRPF LPSPGQFVAW CREEASVTAG LPNVSELVDM VYEYCRKRGL YPDAESYPWK SNAHYWLVTN L YQNMRANA ...String:
MENIAAQMVN FDREQMRRIA NNMPEQYDEK PQVQQVAQII NGVFSQLLAT FPASLANRDQ NEVNEIRRQW VLAFRENGIT TMEQVNAGM RVARRQNRPF LPSPGQFVAW CREEASVTAG LPNVSELVDM VYEYCRKRGL YPDAESYPWK SNAHYWLVTN L YQNMRANA LTDAELRRKA ADELVHMTAR INRGEAIPEP VKQLPVMGGR PLNRAQALAK IAEIKAKFGL KGASV

UniProtKB: Helicase loader

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Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 5 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 5 / Formula: MG
Molecular weightTheoretical: 24.305 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

Concentration4.98 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H17N2NaO4SHEPES-Sodium salt
450.0 mMNaClSodium Chloride
2.0 mMC4H10O2S2Dithiothreitol (DTT)
0.5 mMMgCl2Magnesium Chloride
0.2 mMC10H16N5O13P3Adenosine triphosphate (ATP)
0.25 %C3H8O3Glycerol

Details: 20 mM Na-HEPES pH 7.5, 450mM NaCl, 2mM DTT, 0.5mM MgCl2, 0.2mM ATP, 0.25% Glycerol
GridModel: Quantifoil R0.6/1 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 120 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: Protein BP (1.5 uM) and DNA (1.875 uM) was mixed in a 1.25 molar excess. 3uL of the sample was added to a plasma-cleaned grid at 4 degrees celsius, 100 percent humidity, blot force 4, blot ...Details: Protein BP (1.5 uM) and DNA (1.875 uM) was mixed in a 1.25 molar excess. 3uL of the sample was added to a plasma-cleaned grid at 4 degrees celsius, 100 percent humidity, blot force 4, blot time 4s, wait time 30s, total blots 1, and plunge-frozen into liquid nitrogen-cooled ethane..

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 25595 / Average exposure time: 2.0 sec. / Average electron dose: 51.01 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000
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

Particle selectionNumber selected: 12437695
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

1b79

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.84 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 1137545
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

Initial model
PDB IDChain

1b79

chain_id: A, residue_range: 16-127, source_name: PDB, initial_model_type: experimental model
chain_id: A, residue_range: 128-471, source_name: AlphaFold, initial_model_type: in silico model
chain_id: A, residue_range: 2-118, source_name: AlphaFold, initial_model_type: in silico model

8v9s

chain_id: A, residue_range: 119-192, source_name: PDB, initial_model_type: experimental model
chain_id: A, residue_range: 193-233, source_name: AlphaFold, initial_model_type: in silico model
RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-8v9t:
Ecoli DnaB helicase and Phage Lambda loader P with ADP-Mg in a 6:5 stoichiometry ratio

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