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- PDB-8c5y: RPA tetrameric supercomplex from Pyrococcus abyssi -

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Basic information

Entry
Database: PDB / ID: 8c5y
TitleRPA tetrameric supercomplex from Pyrococcus abyssi
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 / DNA replication / single strand DNA-binding protein / RPA
Function / homology
Function and homology information


nucleic acid binding / chromosome, telomeric region / DNA binding / metal ion 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 (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.35 Å
AuthorsMadru, C. / Martinez-Carranza, M. / Legrand, P. / 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
DepositionJan 10, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 3, 2023Provider: repository / Type: Initial release
Revision 1.1Jul 24, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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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
E: RPA32 subunit of the hetero-oligomeric complex involved in homologous recombination
F: RPA14 subunit of the hetero-oligomeric complex involved in homologous recombination
G: 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
J: Replication factor A
K: RPA32 subunit of the hetero-oligomeric complex involved in homologous recombination
L: RPA14 subunit of the hetero-oligomeric complex involved in homologous recombination
hetero molecules


Theoretical massNumber of molelcules
Total (without water)217,23616
Polymers216,97412
Non-polymers2624
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Replication factor A


Mass: 20234.057 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus abyssi (archaea) / Strain: GE5 / ORSAY / 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: 20930.475 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus abyssi (archaea) / Strain: GE5 / ORSAY / 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: 13078.994 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus abyssi (archaea) / Strain: GE5 / ORSAY / Gene: PAB2164 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9V1Z0
#4: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn
Has ligand of interestN

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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

ComponentName: RPA tetrameric supercomplex / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.345 MDa / Experimental value: YES
Source (natural)Organism: Pyrococcus abyssi (archaea)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
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

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2800 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
4cryoSPARC3.3.2CTF correction
10cryoSPARC3.3.2initial Euler assignment
11cryoSPARC3.3.2final Euler assignment
12cryoSPARC3.3.2classification
13cryoSPARC3.3.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.35 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 141325 / Symmetry type: POINT
RefinementHighest resolution: 3.35 Å

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