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- PDB-8aaj: Crystal structure of the Pyrococcus abyssi RPA (apo form) -

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Basic information

Entry
Database: PDB / ID: 8aaj
TitleCrystal structure of the Pyrococcus abyssi RPA (apo form)
Components
  • RPA14 subunit of the hetero-oligomeric complex involved in homologous recombination
  • RPA32 subunit of the hetero-oligomeric complex involved in homologous recombination
  • Replication factor A
KeywordsDNA BINDING PROTEIN / Replication protein A / ssDNA-Binding protein
Function / homology
Function and homology information


macromolecule metabolic process / intracellular organelle / primary metabolic process / : / nucleic acid binding / DNA binding
Similarity search - Function
Replication factor A protein-like / 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 / Winged helix-like DNA-binding domain superfamily / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
Replication factor A / RPA14 subunit of the hetero-oligomeric complex involved in homologous recombination / RPA32 subunit of the hetero-oligomeric complex involved in homologous recombination
Similarity search - Component
Biological speciesPyrococcus abyssi GE5 (archaea)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 3.7 Å
AuthorsLegrand, P. / Madru, C. / 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
DepositionJul 1, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 3, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Replication factor A
B: RPA32 subunit of the hetero-oligomeric complex involved in homologous recombination
C: RPA14 subunit of the hetero-oligomeric complex involved in homologous recombination
hetero molecules


Theoretical massNumber of molelcules
Total (without water)87,51615
Polymers86,3943
Non-polymers1,12212
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area8090 Å2
ΔGint-163 kcal/mol
Surface area33390 Å2
MethodPISA
Unit cell
Length a, b, c (Å)116.526, 169.738, 201.855
Angle α, β, γ (deg.)90, 90, 90
Int Tables number20
Space group name H-MC2221

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Components

#1: Protein Replication factor A


Mass: 41008.965 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Mutation T2S from reference sequence GenBank: CCE69663.1 to improve expression
Source: (gene. exp.) Pyrococcus abyssi GE5 (archaea) / Strain: GE5 / Orsay / Gene: PAB2163 / Plasmid: pRSFduet(+) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: G8ZHS0
#2: Protein RPA32 subunit of the hetero-oligomeric complex involved in homologous recombination


Mass: 31376.152 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Mutation of SER after first M from reference sequence NCBI Reference Sequence: WP_048146526.1 to improve expression
Source: (gene. exp.) Pyrococcus abyssi GE5 (archaea) / Strain: GE5 / Orsay / Gene: PAB2165 / Plasmid: pRSFduet(+) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9V1Z1
#3: Protein RPA14 subunit of the hetero-oligomeric complex involved in homologous recombination


Mass: 14008.925 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus abyssi GE5 (archaea) / Strain: GE5 / Orsay / Gene: PAB2164 / Plasmid: pRSFduet(+) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9V1Z0
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: SO4
Has ligand of interestY

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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: 5.78 Å3/Da / Density % sol: 78.71 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop / pH: 8 / Details: 0.1M Tris pH 8.5 1.2M ammonium sulfate

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SOLEIL / Beamline: PROXIMA 1 / Wavelength: 0.978565 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Mar 28, 2021 / Details: KB Mirrors
RadiationMonochromator: Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.978565 Å / Relative weight: 1
ReflectionResolution: 3.7→49.35 Å / Num. obs: 13191 / % possible obs: 60.1 % / Redundancy: 27.6 % / Biso Wilson estimate: 166.58 Å2 / CC1/2: 1 / Rmerge(I) obs: 0.082 / Rpim(I) all: 0.016 / Rrim(I) all: 0.083 / Net I/σ(I): 21.4
Reflection shellResolution: 3.7→3.8 Å / Redundancy: 26.8 % / Rmerge(I) obs: 4.306 / Mean I/σ(I) obs: 1 / Num. unique obs: 217 / CC1/2: 0.542 / Rpim(I) all: 0.844 / Rrim(I) all: 4.39 / % possible all: 13.3

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Processing

Software
NameVersionClassification
BUSTER2.10.4 (3-FEB-2022)refinement
XDSVERSION Feb 5, 2021data reduction
STARANISO2.3.36data scaling
SHELXDphasing
PHASERphasing
PARROTphasing
MxCuBE2.1data collection
Coot0.9.8.1model building
RefinementMethod to determine structure: SAD / Resolution: 3.7→49.35 Å / Cor.coef. Fo:Fc: 0.945 / Cor.coef. Fo:Fc free: 0.935 / Cross valid method: THROUGHOUT / SU Rfree Blow DPI: 0.689
RfactorNum. reflection% reflectionSelection details
Rfree0.2547 1036 -RANDOM
Rwork0.2377 ---
obs0.239 13191 60.8 %-
Displacement parametersBiso mean: 235.87 Å2
Baniso -1Baniso -2Baniso -3
1--16.2342 Å20 Å20 Å2
2--2.5722 Å20 Å2
3---13.6621 Å2
Refine analyzeLuzzati coordinate error obs: 0.64 Å
Refinement stepCycle: LAST / Resolution: 3.7→49.35 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5301 0 56 0 5357
Refine LS restraints
Refine-IDTypeDev idealNumberRestraint functionWeight
X-RAY DIFFRACTIONt_bond_d0.0045424HARMONIC2
X-RAY DIFFRACTIONt_angle_deg0.637307HARMONIC2
X-RAY DIFFRACTIONt_dihedral_angle_d2023SINUSOIDAL2
X-RAY DIFFRACTIONt_gen_planes907HARMONIC5
X-RAY DIFFRACTIONt_it5424HARMONIC10
X-RAY DIFFRACTIONt_chiral_improper_torsion698SEMIHARMONIC5
X-RAY DIFFRACTIONt_utility_distance4HARMONIC1
X-RAY DIFFRACTIONt_utility_angle6HARMONIC1
X-RAY DIFFRACTIONt_ideal_dist_contact3590SEMIHARMONIC4
X-RAY DIFFRACTIONt_omega_torsion1.66
X-RAY DIFFRACTIONt_other_torsion13.64
LS refinement shellResolution: 3.7→3.81 Å
RfactorNum. reflection% reflection
Rfree0.311 24 -
Rwork0.3485 --
obs0.345 264 14.12 %
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)
18.31552.9104-2.91048.31551.25598.3154-0.0889-0.4887-0.5221-0.48870.4158-0.4575-0.5221-0.4575-0.3269-0.1112-0.13320.1520.3001-0.1520.053479.4419108.643-7.411
26.1352.91042.91048.31552.91048.3154-0.2028-0.5442-0.5442-0.54420.34140.5442-0.54420.5442-0.13870.10390.0521-0.1520.1653-0.1520.205178.121874.5765-13.1683
36.158-2.1624-0.11828.31552.91046.88570.03190.54420.20050.54420.21210.54420.20050.5442-0.244-0.3040.152-0.1307-0.304-0.128-0.30465.702752.9917-24.3591
44.4761-0.1838-1.72624.6969-0.90527.7582-0.2312-0.5442-0.2884-0.54420.2227-0.5442-0.2884-0.54420.0085-0.304-0.152-0.01310.1514-0.049-0.26344.460950.0178-41.576
58.31551.9221-2.6113.4201-2.64776.6827-0.3336-0.4510.5442-0.4510.2048-0.54420.5442-0.54420.12880.0496-0.1520.1520.304-0.1520.118932.375632.181-26.4531
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1{ A|2 - A|58 }A2 - 58
2X-RAY DIFFRACTION2{ A|59 - A|185 }A59 - 185
3X-RAY DIFFRACTION3{ A|186 - A|401 }A186 - 358
4X-RAY DIFFRACTION3{ A|186 - A|401 }A401
5X-RAY DIFFRACTION4{ B|* }B1 - 185
6X-RAY DIFFRACTION5{ C|* }C6 - 117

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