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- PDB-9f28: Crystal structure of the heterodimeric primase from pyrococcus ab... -

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Basic information

Entry
Database: PDB / ID: 9f28
TitleCrystal structure of the heterodimeric primase from pyrococcus abyssi (deletion of the PriL-CTD domain)
Components
  • DNA primase large subunit PriL
  • DNA primase small subunit PriS
KeywordsREPLICATION / DNA primase / PriS / PriL
Function / homology
Function and homology information


primosome complex / : / DNA-directed RNA polymerase complex / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / 4 iron, 4 sulfur cluster binding / metal ion binding
Similarity search - Function
DNA primase large subunit PriL / DNA primase small subunit PriS / DNA primase, small subunit, eukaryotic/archaeal / DNA primase, small subunit / DNA primase small subunit / DNA primase large subunit, eukaryotic/archaeal / Eukaryotic and archaeal DNA primase, large subunit
Similarity search - Domain/homology
FORMIC ACID / DNA primase large subunit PriL / DNA primase small subunit PriS
Similarity search - Component
Biological speciesPyrococcus abyssi (archaea)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.85 Å
AuthorsMadru, C. / Sauguet, L.
Funding support France, 1items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ARCHAPOL France
CitationJournal: Nat Commun / Year: 2024
Title: Communication between DNA polymerases and Replication Protein A within the archaeal replisome.
Authors: Markel Martínez-Carranza / Léa Vialle / Clément Madru / Florence Cordier / Ayten Dizkirici Tekpinar / Ahmed Haouz / Pierre Legrand / Rémy A Le Meur / Patrick England / Rémi Dulermo / J ...Authors: Markel Martínez-Carranza / Léa Vialle / Clément Madru / Florence Cordier / Ayten Dizkirici Tekpinar / Ahmed Haouz / Pierre Legrand / Rémy A Le Meur / Patrick England / Rémi Dulermo / J Iñaki Guijarro / Ghislaine Henneke / Ludovic Sauguet /
Abstract: Replication Protein A (RPA) plays a pivotal role in DNA replication by coating and protecting exposed single-stranded DNA, and acting as a molecular hub that recruits additional replication factors. ...Replication Protein A (RPA) plays a pivotal role in DNA replication by coating and protecting exposed single-stranded DNA, and acting as a molecular hub that recruits additional replication factors. We demonstrate that archaeal RPA hosts a winged-helix domain (WH) that interacts with two key actors of the replisome: the DNA primase (PriSL) and the replicative DNA polymerase (PolD). Using an integrative structural biology approach, combining nuclear magnetic resonance, X-ray crystallography and cryo-electron microscopy, we unveil how RPA interacts with PriSL and PolD through two distinct surfaces of the WH domain: an evolutionarily conserved interface and a novel binding site. Finally, RPA is shown to stimulate the activity of PriSL in a WH-dependent manner. This study provides a molecular understanding of the WH-mediated regulatory activity in central replication factors such as RPA, which regulate genome maintenance in Archaea and Eukaryotes.
History
DepositionApr 22, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 4, 2024Provider: repository / Type: Initial release
Revision 1.1Jan 22, 2025Group: Data collection / Database references / Category: citation / citation_author / diffrn
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name / _diffrn.ambient_temp

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA primase small subunit PriS
B: DNA primase large subunit PriL
hetero molecules


Theoretical massNumber of molelcules
Total (without water)67,4037
Polymers67,1972
Non-polymers2065
Water11,007611
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2550 Å2
ΔGint-26 kcal/mol
Surface area25350 Å2
MethodPISA
Unit cell
Length a, b, c (Å)116.574, 116.574, 121.084
Angle α, β, γ (deg.)90, 90, 90
Int Tables number96
Space group name H-MP43212

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein DNA primase small subunit PriS / DNA primase 41 kDa subunit / Pabp41 / p41


Mass: 41049.980 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus abyssi (archaea) / Gene: priS, priA, PYRAB01820, PAB2236 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q9V292, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases
#2: Protein DNA primase large subunit PriL / DNA primase 46 kDa subunit / Pabp46 / p46


Mass: 26147.143 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus abyssi (archaea) / Gene: priL, priB, PYRAB01830, PAB2235 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9V291

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Non-polymers , 4 types, 616 molecules

#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#5: Chemical ChemComp-FMT / FORMIC ACID


Mass: 46.025 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: CH2O2
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 611 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN
Has protein modificationN

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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: 3.06 Å3/Da / Density % sol: 59.82 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop / Details: 0.2M Magnesium Formate

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SOLEIL / Beamline: PROXIMA 2 / Wavelength: 0.98011 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Mar 24, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.98011 Å / Relative weight: 1
ReflectionResolution: 1.85→39.51 Å / Num. obs: 67117 / % possible obs: 100 % / Redundancy: 26.9 % / CC1/2: 0.999 / Rmerge(I) obs: 0.114 / Rpim(I) all: 0.023 / Net I/σ(I): 16.4
Reflection shellResolution: 1.85→1.89 Å / Rmerge(I) obs: 2.08 / Mean I/σ(I) obs: 1.6 / Num. unique obs: 4200 / CC1/2: 0.592 / Rpim(I) all: 0.609

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Processing

Software
NameVersionClassification
BUSTER2.10.4refinement
XDSdata reduction
Aimlessdata scaling
AutoSolphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.85→39.51 Å / Cor.coef. Fo:Fc: 0.953 / Cor.coef. Fo:Fc free: 0.949 / SU R Cruickshank DPI: 0.126 / Cross valid method: THROUGHOUT / SU R Blow DPI: 0.136 / SU Rfree Blow DPI: 0.123 / SU Rfree Cruickshank DPI: 0.118
RfactorNum. reflection% reflectionSelection details
Rfree0.2236 3348 -RANDOM
Rwork0.1987 ---
obs0.1999 67117 93.4 %-
Displacement parametersBiso mean: 40.81 Å2
Baniso -1Baniso -2Baniso -3
1-0.6295 Å20 Å20 Å2
2--0.6295 Å20 Å2
3----1.2589 Å2
Refine analyzeLuzzati coordinate error obs: 0.23 Å
Refinement stepCycle: LAST / Resolution: 1.85→39.51 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4606 0 9 611 5226
Refine LS restraints
Refine-IDTypeDev idealNumberRestraint functionWeight
X-RAY DIFFRACTIONt_bond_d0.0094716HARMONIC2
X-RAY DIFFRACTIONt_angle_deg0.896338HARMONIC2
X-RAY DIFFRACTIONt_dihedral_angle_d1739SINUSOIDAL2
X-RAY DIFFRACTIONt_gen_planes798HARMONIC5
X-RAY DIFFRACTIONt_it4716HARMONIC10
X-RAY DIFFRACTIONt_chiral_improper_torsion580SEMIHARMONIC5
X-RAY DIFFRACTIONt_utility_distance4HARMONIC1
X-RAY DIFFRACTIONt_ideal_dist_contact4397SEMIHARMONIC4
X-RAY DIFFRACTIONt_omega_torsion3.3
X-RAY DIFFRACTIONt_other_torsion17.4
LS refinement shellResolution: 1.85→1.89 Å
RfactorNum. reflection% reflection
Rfree0.2494 59 -
Rwork0.2557 --
obs0.2554 1343 26.87 %
Refinement TLS params.

Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.462-0.6618-0.01380.72510.60520.91820.12650.05630.15080.0563-0.2096-0.32610.1508-0.32610.0832-0.029-0.0677-0.05430.08070.0068-0.03063.9792-37.233528.5735
20.67460.20071.38360.65360.61652.55680.25610.20320.07340.2032-0.0588-0.45480.0734-0.4548-0.19730.0414-0.0244-0.03230.0363-0.0133-0.041913.6857-9.890259.3333
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1{ A|* }A1 - 344
2X-RAY DIFFRACTION1{ A|* }A350 - 352
3X-RAY DIFFRACTION2{ B|* }B1 - 211

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