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- EMDB-70271: Ecoli DnaB helicase and Phage Lambda loader P with ADP-Mg in a 6:... -

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Basic information

Entry
Database: EMDB / ID: EMD-70271
TitleEcoli DnaB helicase and Phage Lambda loader P with ADP-Mg in a 6:6 stoichiometry ratio.
Map data
Sample
  • Complex: Replication initiation intermediate with Escherichia coli DnaB helicase and Phage Lambda loader P in a 6:6 stoichiometry ratio
    • Complex: Ecoli DnaB helicase
      • Protein or peptide: Replicative DNA helicase
    • Complex: Phage Lambda loader P
      • Protein or peptide: Helicase loader
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
KeywordsHexameric DnaB helicase / Phage Lambda P helicase loader / bacterial DNA replication initiation intermediate / 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: 3.85 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: bioRxiv / 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 require loader proteins for assembly at the origins of DNA replication. Multiple copies of the bacteriophage λP (P) loader bind to and load the DnaB (B) replicative helicase ...Replicative helicases require loader proteins for assembly at the origins of DNA replication. Multiple copies of the bacteriophage λP (P) loader bind to and load the DnaB (B) replicative helicase on replication-origin-derived single-stranded DNA. We find that the DnaB•λP complex exists in two forms: B P and B P . In the 2.66 Å cryo-EM model of B P , five copies of the λP loader assemble into a crown-like shape that tightly grips DnaB. In this complex, closed planar DnaB is reconfigured into an open spiral with a sufficiently sized breach to permit ssDNA to enter an internal chamber. The transition to the open spiral involves λP-mediated changes to the Docking Helix (DH)-Linker Helix (LH) interface. The loader directly stabilizes the open spiral. Unexpectedly, one λP chain in B P is bound across the breach, precluding entry of replication-origin-derived ssDNA into DnaB's central chamber. We suggest that the B P complex is an early intermediate in the helicase activation pathway wherein neither the DnaB helicase nor the λP loader has attained its final form. DnaB in this complex adopts a partially open planar configuration, termed ajar planar. The partially ordered λP loader assembly features a much looser interaction with DnaB. The ssDNA and ATP sites in both complexes are in a configuration ill-suited for binding or hydrolysis. Our work specifies the conformational changes required for the intermediate B P to transition to B P on the pathway to recruitment by the initiator protein complex to the replication origin.
#1: Journal: Biorxiv / Year: 2024
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
DepositionApr 18, 2025-
Header (metadata) releaseJul 16, 2025-
Map releaseJul 16, 2025-
UpdateJul 16, 2025-
Current statusJul 16, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_70271.png

Downloads & links

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Map

FileDownload / File: emd_70271.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 400 pix.
= 433.2 Å		1.08 Å/pix.
x 400 pix.
= 433.2 Å		1.08 Å/pix.
x 400 pix.
= 433.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.083 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.109
Minimum - Maximum-0.001793018 - 2.3803186
Average (Standard dev.)0.0005537458 (±0.016108785)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 433.19998 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_70271_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_70271_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 : Replication initiation intermediate with Escherichia coli DnaB he...

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

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Supramolecule #1: Replication initiation intermediate with Escherichia coli DnaB he...

SupramoleculeName: Replication initiation intermediate with Escherichia coli DnaB helicase and Phage Lambda loader P in a 6:6 stoichiometry ratio
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Molecular weightTheoretical: 473.4 KDa

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Supramolecule #2: Ecoli DnaB helicase

SupramoleculeName: Ecoli DnaB helicase / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Escherichia coli (E. coli)

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Supramolecule #3: Phage Lambda loader P

SupramoleculeName: Phage Lambda loader P / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Escherichia phage Lambda (virus)

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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: 6 / 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: 6 / 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: 6 / 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

Concentration4.9 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMNaC8H17N2O4SHEPES-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), ssDNA (1.875 uM), and LO-156-299-NHis (2.25 uM) was mixed at 1.25 and 1.5 molar excess, respectively. 3uL of the sample was added to a plasma-cleaned grid at 4 degrees ...Details: Protein BP (1.5 uM), ssDNA (1.875 uM), and LO-156-299-NHis (2.25 uM) was mixed at 1.25 and 1.5 molar excess, respectively. 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
SoftwareName: Leginon
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 6479 / Average exposure time: 2.0 sec. / Average electron dose: 51.19 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: 2.7 µm / Nominal defocus min: 0.6 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: 2315940
Details: Particle picking was performed using Topaz in CryoSPARC.
CTF correctionSoftware - Name: cryoSPARC (ver. 4) / Type: 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: 3.85 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4) / Number images used: 572557
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

1b79

chain_id: A, residue_range: 24-127, 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
SoftwareName: PHENIX (ver. 1.21.2_5419)
RefinementSpace: REAL / Protocol: OTHER / Overall B value: 153.98
Output model

PDB-9oa2:
Ecoli DnaB helicase and Phage Lambda loader P with ADP-Mg in a 6:6 stoichiometry ratio.

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