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- PDB-8v9s: Distinct Quaternary States, Intermediates, and Autoinhibition Dur... -

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Entry
Database: PDB / ID: 8v9s
TitleDistinct Quaternary States, Intermediates, and Autoinhibition During Loading of the DnaB-Replicative Helicase by the Phage Lambda P Helicase Loader
ComponentsReplication protein P
KeywordsREPLICATION / hexameric DnaB helicase / replicative loader / Phage Lambda P helicase loader / bacterial DNA replication initiation
Function / homologyReplication P / Replication protein P / bidirectional double-stranded viral DNA replication / DNA replication initiation / Helicase loader
Function and homology information
Biological speciesEscherichia phage Lambda (virus)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 1.86 Å
AuthorsBrown, D. / Shatarupa, A. / Olinares, P.D.B. / Chase, J. / Isiorho, E. / Chait, B. / Jeruzalmi, D.
Funding support United States, 1items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)1818255 United States
CitationJournal: 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.
History
DepositionDec 9, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 16, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Replication protein P


Theoretical massNumber of molelcules
Total (without water)13,4051
Polymers13,4051
Non-polymers00
Water1,36976
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)49.731, 49.731, 70.772
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number152
Space group name H-MP3121
Space group name HallP312"
Symmetry operation#1: x,y,z
#2: -y,x-y,z+1/3
#3: -x+y,-x,z+2/3
#4: x-y,-y,-z+2/3
#5: -x,-x+y,-z+1/3
#6: y,x,-z

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Components

#1: Protein Replication protein P


Mass: 13405.462 Da / Num. of mol.: 1 / Fragment: residues 105 to 210
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia phage Lambda (virus) / Plasmid: pCDFDuet
Details (production host): Duet expression system; Streptomycin resistance
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P03689
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 76 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 1.92 Å3/Da / Density % sol: 35.97 %
Description: Smooth rhombus; Trigonal pyramidal with one screw.
Crystal growTemperature: 298.15 K / Method: vapor diffusion, sitting drop
Details: Crystals were prepared using the sitting drop vapor diffusion method by mixing either 0.1, or 0.2, or 0.4 uL of the protein solution and 0.2 uL of a series of commercially available ...Details: Crystals were prepared using the sitting drop vapor diffusion method by mixing either 0.1, or 0.2, or 0.4 uL of the protein solution and 0.2 uL of a series of commercially available crystallization screens (Qiagen). Optimized crystals for X-ray diffraction were grown at room temperature in a buffer consisting of 3 M to 4 M NaCl with 0.1 M HEPES-NaOH, pH 7 to 8 and required 7 days to form.
PH range: 7 - 8

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Data collection

DiffractionMean temperature: 100 K / Ambient temp details: cold nitrogen gas stream / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.97918 Å
DetectorType: DECTRIS EIGER2 X 16M / Detector: PIXEL / Date: Jun 30, 2021
RadiationMonochromator: dual-canted undulatory / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97918 Å / Relative weight: 1
ReflectionResolution: 1.86→43.07 Å / Num. obs: 8852 / % possible obs: 98.49 % / Redundancy: 5.7 % / Biso Wilson estimate: 20.16 Å2 / CC1/2: 0.987 / CC star: 0.997 / Rpim(I) all: 0.047 / Rrim(I) all: 0.115 / Rsym value: 0.105 / Χ2: 0.824 / Net I/av σ(I): 18.98 / Net I/σ(I): 18.99
Reflection shellResolution: 1.86→1.98 Å / Mean I/σ(I) obs: 0.8 / Num. unique obs: 1205 / CC1/2: 0.678 / CC star: 0.899 / Rpim(I) all: 0.364 / Rrim(I) all: 0.721 / Χ2: 0.251

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Processing

Software
NameVersionClassification
PHENIX1.20.1-4487-000refinement
PHENIX1.20.1-4487-000model building
HKL-2000v721.2data reduction
HKL-2000v721.2data scaling
PHENIX1.20.1-4487-000phasing
RefinementMethod to determine structure: SAD / Resolution: 1.86→43.07 Å / SU ML: 0.24 / Cross valid method: FREE R-VALUE / σ(F): 0.15 / Phase error: 25.73
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2436 870 9.98 %
Rwork0.2258 7848 -
obs0.2276 8718 98.49 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 27.94 Å2
Refinement stepCycle: LAST / Resolution: 1.86→43.07 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms607 0 0 76 683
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0108621
X-RAY DIFFRACTIONf_angle_d1.256842
X-RAY DIFFRACTIONf_chiral_restr0.052988
X-RAY DIFFRACTIONf_plane_restr0.0142109
X-RAY DIFFRACTIONf_dihedral_angle_d5.155986
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.86-1.980.31551370.29831205X-RAY DIFFRACTION94
1.98-2.130.29811440.22331292X-RAY DIFFRACTION98.76
2.13-2.350.25891460.25331299X-RAY DIFFRACTION98.7
2.35-2.690.25841430.23341301X-RAY DIFFRACTION99.59
2.69-3.390.25411430.2321347X-RAY DIFFRACTION99.67
3.39-43.070.19881570.19681404X-RAY DIFFRACTION100
Refinement TLS params.Method: refined / Origin x: 10.5053836562 Å / Origin y: 29.1086348662 Å / Origin z: 15.0877517953 Å
111213212223313233
T0.256231957948 Å20.192865377138 Å2-0.051313889178 Å2-0.128249385432 Å2-0.0712631144211 Å2--0.0986557014693 Å2
L0.0796461148361 °2-0.00610451244823 °2-0.0242151916152 °2-0.0017721020955 °20.00428897454681 °2--0.00775584809801 °2
S0.0582811732352 Å °0.0376724836247 Å °-0.00435064833951 Å °0.0088133513492 Å °-0.00789480612036 Å °-0.00758404462291 Å °0.00344009132536 Å °0.0228599734257 Å °0.127139026381 Å °
Refinement TLS groupSelection details: all

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