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- PDB-8oej: Extended RPA-DNA nucleoprotein filament -

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Basic information

Entry
Database: PDB / ID: 8oej
TitleExtended RPA-DNA nucleoprotein filament
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
  • poly dT
KeywordsDNA BINDING PROTEIN / DNA replication / single strand DNA-binding protein / RPA
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
DNA / DNA (> 10) / DNA (> 100) / 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 (archaea)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.96 Å
AuthorsMadru, C. / Martinez-Carranza, M. / Sauguet, L.
Funding support France, 1items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR) 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
DepositionMar 10, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 10, 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
D: Replication factor A
H: RPA32 subunit of the hetero-oligomeric complex involved in homologous recombination
I: RPA14 subunit of the hetero-oligomeric complex involved in homologous recombination
T: poly dT
hetero molecules


Theoretical massNumber of molelcules
Total (without water)203,5199
Polymers203,3897
Non-polymers1312
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area18210 Å2
ΔGint-87 kcal/mol
Surface area61490 Å2
MethodPISA

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Components

#1: Protein Replication factor A


Mass: 41008.965 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus abyssi (archaea) / Gene: PAB2163 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: G8ZHS0
#2: Protein RPA32 subunit of the hetero-oligomeric complex involved in homologous recombination


Mass: 31489.309 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus abyssi (archaea) / Gene: PAB2165 / 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.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus abyssi (archaea) / Gene: PAB2164 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9V1Z0
#4: DNA chain poly dT


Mass: 30374.275 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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
1RPA bound to a 100-mer poly-dT ssDNACOMPLEX#1-#40MULTIPLE SOURCES
2RPA 1,2,3COMPLEX#1-#31RECOMBINANT
3poly dTCOMPLEX#41RECOMBINANT
Molecular weightValue: 0.18 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Pyrococcus abyssi (archaea)29292
33Escherichia coli (E. coli)562
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli BL21(DE3) (bacteria)469008
33Synthetic construct (others)32630
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2800 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

Image processing
IDImage recording-ID
11
21
CTF correction
IDEM image processing-IDType
11PHASE FLIPPING AND AMPLITUDE CORRECTION
22PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstruction
IDResolution (Å)Resolution methodNum. of particlesImage processing-IDEntry-IDSymmetry type
17.96FSC 0.143 CUT-OFF3447818OEJPOINT
28FSC 0.143 CUT-OFF3447818OEJPOINT
37.96FSC 0.143 CUT-OFF3447828OEJPOINT
48FSC 0.143 CUT-OFF3447828OEJPOINT
RefinementHighest resolution: 7.96 Å

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