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

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Basic information

Entry
Database: PDB / ID: 8v9t
TitleEcoli DnaB helicase and Phage Lambda loader P with ADP-Mg in a 6:5 stoichiometry ratio
Components
  • Helicase loader
  • Replicative DNA helicase
KeywordsDNA BINDING PROTEIN / Hexameric DnaB helicase / Phage Lambda P helicase loader / bacterial DNA replication initiation / auto inhibition
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
ADENOSINE-5'-DIPHOSPHATE / Helicase loader / Replicative DNA helicase DnaB
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Escherichia phage Lambda (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.84 Å
AuthorsShatarupa, A. / Brown, D. / Olinares, P.D.B. / Chase, J. / Isiorho, E. / Chait, B.T. / Jeruzalmi, D.
Funding support United States, 1items
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, P.D.B. / Chase, J. / Isiorho, E. / Chait, B.T. / Jeruzalmi, D.
History
DepositionDec 9, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 4, 2026Provider: repository / Type: Initial release
Revision 1.0Mar 4, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 4, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 4, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 4, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Mar 4, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Replicative DNA helicase
B: Replicative DNA helicase
C: Replicative DNA helicase
D: Replicative DNA helicase
E: Replicative DNA helicase
F: Replicative DNA helicase
V: Helicase loader
W: Helicase loader
X: Helicase loader
Y: Helicase loader
Z: Helicase loader
hetero molecules


Theoretical massNumber of molelcules
Total (without water)449,72021
Polymers447,46211
Non-polymers2,25810
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Replicative DNA helicase


Mass: 52450.945 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: dnaB, groP, grpA, b4052, JW4012 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0ACB0, DNA helicase
#2: Protein
Helicase loader / Lambda P helicase loader / Replication protein P


Mass: 26551.326 Da / Num. of mol.: 5 / Mutation: K2E
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia phage Lambda (virus) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P03689
#3: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestY
Has protein modificationN

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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: Ecoli DnaB helicase and Phage Lambda loader P with ADP-Mg in a 6:5 stoichiometry ratio.
Type: COMPLEX / Entity ID: #1-#2 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.44694 MDa / Experimental value: YES
Buffer solutionpH: 7.5
Details: 20 mM Na-HEPES pH 7.5, 450mM NaCl, 2mM DTT, 0.5mM MgCl2, 0.2mM ATP, 0.25% Glycerol
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPES-Sodium saltC8H17N2NaO4S1
2450 mMSodium ChlorideNaCl1
32 mMDithiothreitol (DTT)C4H10O2S21
40.5 mMMagnesium ChlorideMgCl21
50.2 mMAdenosine triphosphate (ATP)C10H16N5O13P31
60.25 %GlycerolC3H8O31
SpecimenConc.: 4.98 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
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 imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2 sec. / Electron dose: 51.01 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 25595
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1PHENIX1.21.2_5419model refinement
3Warpparticle selection
7cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 12437695
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.84 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1137545 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Atomic model building

3D fitting-ID: 1 / Chain-ID: A

IDPDB-IDPdb chain-IDAccession codeChain residue rangeInitial refinement model-IDPdb chain residue rangeSource nameType
11B79

1b79

A1B7916-127116-127PDBexperimental model
2128-471AlphaFoldin silico model
32-118AlphaFoldin silico model
48V9S

8v9s

A8V9S119-192119-192PDBexperimental model
5193-233AlphaFoldin silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 90.08 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00326790
ELECTRON MICROSCOPYf_angle_d0.536636255
ELECTRON MICROSCOPYf_chiral_restr0.04134057
ELECTRON MICROSCOPYf_plane_restr0.0054790
ELECTRON MICROSCOPYf_dihedral_angle_d10.139710265

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