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- PDB-8aa9: Crystal structure of the Rpa1 AROD-OB-1 domains -

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Basic information

Entry
Database: PDB / ID: 8aa9
TitleCrystal structure of the Rpa1 AROD-OB-1 domains
ComponentsReplication factor A
KeywordsDNA BINDING PROTEIN / Replication protein A / ssDNA-Binding protein
Function / homology
Function and homology information


response to ionizing radiation / double-strand break repair via homologous recombination / DNA binding / metal ion binding
Similarity search - Function
Replication factor A, C-terminal / Replication factor-A C terminal domain / OB-fold nucleic acid binding domain, AA-tRNA synthetase-type / OB-fold nucleic acid binding domain / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
DI(HYDROXYETHYL)ETHER / Replication factor A
Similarity search - Component
Biological speciesPyrococcus abyssi GE5 (archaea)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.8 Å
AuthorsMadru, C. / Legrand, P. / Sauguet, L.
Funding support France, 2items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-10-JCJC-1501 ARCHPOL France
Agence Nationale de la Recherche (ANR)ANR-20-CE11-0003 ARCHAPRIM France
CitationJournal: Nat Commun / Year: 2023
Title: DNA-binding mechanism and evolution of replication protein A.
Authors: Clément Madru / Markel Martínez-Carranza / Sébastien Laurent / Alessandra C Alberti / Maelenn Chevreuil / Bertrand Raynal / Ahmed Haouz / Rémy A Le Meur / Marc Delarue / Ghislaine ...Authors: Clément Madru / Markel Martínez-Carranza / Sébastien Laurent / Alessandra C Alberti / Maelenn Chevreuil / Bertrand Raynal / Ahmed Haouz / Rémy A Le Meur / Marc Delarue / Ghislaine Henneke / Didier Flament / Mart Krupovic / Pierre Legrand / Ludovic Sauguet /
Abstract: Replication Protein A (RPA) is a heterotrimeric single stranded DNA-binding protein with essential roles in DNA replication, recombination and repair. Little is known about the structure of RPA in ...Replication Protein A (RPA) is a heterotrimeric single stranded DNA-binding protein with essential roles in DNA replication, recombination and repair. Little is known about the structure of RPA in Archaea, the third domain of life. By using an integrative structural, biochemical and biophysical approach, we extensively characterize RPA from Pyrococcus abyssi in the presence and absence of DNA. The obtained X-ray and cryo-EM structures reveal that the trimerization core and interactions promoting RPA clustering on ssDNA are shared between archaea and eukaryotes. However, we also identified a helical domain named AROD (Acidic Rpa1 OB-binding Domain), and showed that, in Archaea, RPA forms an unanticipated tetrameric supercomplex in the absence of DNA. The four RPA molecules clustered within the tetramer could efficiently coat and protect stretches of ssDNA created by the advancing replisome. Finally, our results provide insights into the evolution of this primordial replication factor in eukaryotes.
History
DepositionJun 30, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 3, 2023Provider: repository / Type: Initial release
Revision 1.1May 1, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Replication factor A
B: Replication factor A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)45,2854
Polymers45,1442
Non-polymers1422
Water5,224290
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2260 Å2
ΔGint-10 kcal/mol
Surface area18850 Å2
Unit cell
Length a, b, c (Å)52.754, 83.475, 56.195
Angle α, β, γ (deg.)90, 115.33, 90
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein Replication factor A


Mass: 22571.953 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus abyssi GE5 (archaea) / Strain: GE5 / Orsay / Gene: PAB2163 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: G8ZHS0
#2: Chemical ChemComp-PEG / DI(HYDROXYETHYL)ETHER


Mass: 106.120 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H10O3
#3: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 290 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: 2.74 Å3/Da / Density % sol: 55.12 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop / pH: 8.5 / Details: 0.01M NiCl2 20% w/v PEG MME 2K 0.1M Tris pH 8.5

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SOLEIL / Beamline: PROXIMA 1 / Wavelength: 0.97934 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: May 29, 2021 / Details: KB Mirrors
RadiationMonochromator: Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97934 Å / Relative weight: 1
ReflectionResolution: 1.8→47.68 Å / Num. obs: 37718 / % possible obs: 92.5 % / Redundancy: 7 % / Biso Wilson estimate: 37 Å2 / CC1/2: 1 / Rmerge(I) obs: 0.058 / Rpim(I) all: 0.024 / Rrim(I) all: 0.062 / Net I/σ(I): 15.8
Reflection shellResolution: 1.8→1.85 Å / Redundancy: 7 % / Rmerge(I) obs: 1.667 / Mean I/σ(I) obs: 1.1 / Num. unique obs: 1311 / CC1/2: 0.372 / Rpim(I) all: 0.676 / Rrim(I) all: 1.8 / % possible all: 44.2

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Processing

Software
NameVersionClassification
BUSTER2.10.4 (3-FEB-2022)refinement
XDSBUILT=20220110data reduction
STARANISO2.3.77data scaling
MOLREP11.7.03phasing
MxCuBEv2.1data collection
Coot0.9.8.1model building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: Working model of the full RPA

Resolution: 1.8→32.25 Å / Cor.coef. Fo:Fc: 0.956 / Cor.coef. Fo:Fc free: 0.95 / SU R Cruickshank DPI: 0.138 / Cross valid method: THROUGHOUT / SU R Blow DPI: 0.142 / SU Rfree Blow DPI: 0.124 / SU Rfree Cruickshank DPI: 0.123
RfactorNum. reflection% reflectionSelection details
Rfree0.2284 1857 4.92 %RANDOM
Rwork0.2086 ---
obs0.2095 37716 92.4 %-
Displacement parametersBiso mean: 39.74 Å2
Baniso -1Baniso -2Baniso -3
1--0.0597 Å20 Å20.4556 Å2
2---1.2775 Å20 Å2
3---1.3372 Å2
Refine analyzeLuzzati coordinate error obs: 0.26 Å
Refinement stepCycle: LAST / Resolution: 1.8→32.25 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2860 0 8 290 3158
Refine LS restraints
Refine-IDTypeDev idealNumberRestraint functionWeight
X-RAY DIFFRACTIONt_bond_d0.0082967HARMONIC2
X-RAY DIFFRACTIONt_angle_deg0.954012HARMONIC2
X-RAY DIFFRACTIONt_dihedral_angle_d1127SINUSOIDAL2
X-RAY DIFFRACTIONt_gen_planes501HARMONIC5
X-RAY DIFFRACTIONt_it2967HARMONIC10
X-RAY DIFFRACTIONt_chiral_improper_torsion384SEMIHARMONIC5
X-RAY DIFFRACTIONt_sum_occupancies4HARMONIC1
X-RAY DIFFRACTIONt_ideal_dist_contact2914SEMIHARMONIC4
X-RAY DIFFRACTIONt_omega_torsion3.49
X-RAY DIFFRACTIONt_other_torsion14.32
LS refinement shellResolution: 1.8→1.83 Å
RfactorNum. reflection% reflection
Rfree0.3423 26 3.4 %
Rwork0.3088 729 -
obs--36.75 %
Refinement TLS params.Origin x: 27.171 Å / Origin y: 45.6439 Å / Origin z: 0.8697 Å
111213212223313233
T-0.001 Å20.0089 Å2-0.0009 Å2--0.0416 Å20.0133 Å2--0.0111 Å2
L0.5985 °2-0.1175 °2-0.1837 °2-0.0133 °20.0567 °2---0.0148 °2
S0.0015 Å °-0.0447 Å °-0.0215 Å °-0.0447 Å °-0.0125 Å °0.013 Å °-0.0215 Å °0.013 Å °0.011 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1{ *|* }A3 - 180
2X-RAY DIFFRACTION1{ *|* }A201
3X-RAY DIFFRACTION1{ *|* }B2 - 180
4X-RAY DIFFRACTION1{ *|* }C1
5X-RAY DIFFRACTION1{ *|* }S1 - 290

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