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

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Basic information

Entry
Database: PDB / ID: 9oa2
TitleEcoli DnaB helicase and Phage Lambda loader P with ADP-Mg in a 6:6 stoichiometry ratio.
Components
  • Helicase loader
  • Replicative DNA helicase
KeywordsDNA BINDING PROTEIN / Hexameric DnaB helicase / Phage Lambda P helicase loader / bacterial DNA replication initiation intermediate
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: 3.85 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: 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, P.D.B. / Chase, J. / Isiorho, E. / Chait, B.T. / Jeruzalmi, D.
History
DepositionApr 18, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 16, 2025Provider: 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
U: Helicase loader
V: Helicase loader
W: Helicase loader
X: Helicase loader
Y: Helicase loader
Z: Helicase loader
hetero molecules


Theoretical massNumber of molelcules
Total (without water)476,72324
Polymers474,01412
Non-polymers2,70912
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: mass spectrometry, 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.: 6 / 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.: 6 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
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

Component
IDNameTypeEntity IDParent-IDSource
1Replication initiation intermediate with Escherichia coli DnaB helicase and Phage Lambda loader P in a 6:6 stoichiometry ratioCOMPLEX#1-#20MULTIPLE SOURCES
2Ecoli DnaB helicaseCOMPLEX#11RECOMBINANT
3Phage Lambda loader PCOMPLEX#21RECOMBINANT
Molecular weightValue: 0.4734 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (E. coli)562
23Escherichia phage Lambda (virus)2681611
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli BL21(DE3) (bacteria)469008
23Escherichia coli BL21(DE3) (bacteria)469008
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 saltNaC8H17N2O4S1
2450 mMSodium ChlorideNaCl1
32 mMDithiothreitol (DTT)C4H10O2S21
40.5 mMMagnesium ChlorideMgCl21
50.2 mMAdenosine triphosphate (ATP)C10H16N5O13P31
60.25 %GlycerolC3H8O31
SpecimenConc.: 4.9 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), 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 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: 2700 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2 sec. / Electron dose: 51.19 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6479
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4particle selection
2Leginonimage acquisition
4cryoSPARC4CTF correction
7PHENIX1.21.2_5419model fitting
12cryoSPARC43D reconstruction
13PHENIX1.21.2_5419model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2315940
Details: Particle picking was performed using Topaz in CryoSPARC.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.85 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 572557 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingB value: 153.98 / Protocol: 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

A1B7924-127124-127PDBexperimental model
2128-471AlphaFoldin silico model
32-118AlphaFoldin silico model
48V9S

8v9s

A8V9S119-1923119-192PDBexperimental model
5193-233AlphaFoldin silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 153.99 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002525320
ELECTRON MICROSCOPYf_angle_d0.578534259
ELECTRON MICROSCOPYf_chiral_restr0.04063851
ELECTRON MICROSCOPYf_plane_restr0.00454510
ELECTRON MICROSCOPYf_dihedral_angle_d9.31079701

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