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- EMDB-16444: RPA tetrameric supercomplex from Pyrococcus abyssi -

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Basic information

Entry
Database: EMDB / ID: EMD-16444
TitleRPA tetrameric supercomplex from Pyrococcus abyssi
Map data
Sample
  • Complex: RPA tetrameric supercomplex
    • Protein or peptide: Replication factor A
    • Protein or peptide: RPA32 subunit of the hetero-oligomeric complex involved in homologous recombination
    • Protein or peptide: RPA14 subunit of the hetero-oligomeric complex involved in homologous recombination
  • Ligand: ZINC ION
KeywordsDNA replication / single strand DNA-binding protein / RPA / DNA 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 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.35 Å
AuthorsMadru C / Martinez-Carranza M / Legrand P / Sauguet L
Funding support France, 1 items
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, 2023-
Header (metadata) releaseMay 3, 2023-
Map releaseMay 3, 2023-
UpdateJul 24, 2024-
Current statusJul 24, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16444.png

Downloads & links

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Map

FileDownload / File: emd_16444.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 384 pix.
= 330.24 Å		0.86 Å/pix.
x 384 pix.
= 330.24 Å		0.86 Å/pix.
x 384 pix.
= 330.24 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.86 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.145
Minimum - Maximum-0.9540051 - 1.4544634
Average (Standard dev.)-0.00023487801 (±0.023180647)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 330.24 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16444_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_16444_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_16444_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : RPA tetrameric supercomplex

EntireName: RPA tetrameric supercomplex
Components
  • Complex: RPA tetrameric supercomplex
    • Protein or peptide: Replication factor A
    • Protein or peptide: RPA32 subunit of the hetero-oligomeric complex involved in homologous recombination
    • Protein or peptide: RPA14 subunit of the hetero-oligomeric complex involved in homologous recombination
  • Ligand: ZINC ION

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Supramolecule #1: RPA tetrameric supercomplex

SupramoleculeName: RPA tetrameric supercomplex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Pyrococcus abyssi (archaea)
Molecular weightTheoretical: 345 KDa

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Macromolecule #1: Replication factor A

MacromoleculeName: Replication factor A / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Pyrococcus abyssi (archaea) / Strain: GE5 / ORSAY
Molecular weightTheoretical: 20.234057 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
VATYTRKKIK DIEAGDRFVE VRGTIAKVYR VLTYDACPEC KKKVDYDEGL GVWICPEHGE VQPIKMTILD FGLDDGTGYI RVTLFGDDA EELLGVSPEE IAEKIKELEE SGLTTKEAAR KLAEDEFYNI IGREIVVRGN VIEDRFLGLI LRASSWEDVD Y RREIERIK EELEKLGVM

UniProtKB: Replication factor A

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Macromolecule #2: RPA32 subunit of the hetero-oligomeric complex involved in homolo...

MacromoleculeName: RPA32 subunit of the hetero-oligomeric complex involved in homologous recombination
type: protein_or_peptide / ID: 2 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Pyrococcus abyssi (archaea) / Strain: GE5 / ORSAY
Molecular weightTheoretical: 20.930475 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
KKRMPATRLY IKDILEGYFV KSEGDFEPNY LITKYARKVY RAKIVGTVVR EPLIAEDETY GKFQVDDGTG VIWVLGFRDD TKFAKLVRK GDLVQVIGKI AEWRDDKQIL VEGVSKVHPN MWILHRYETL KEKIEHIKKA KIALEIYNQY GITAKSKVIA K NKGIEEEL LEVIDELYGI MM

UniProtKB: RPA32 subunit of the hetero-oligomeric complex involved in homologous recombination

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Macromolecule #3: RPA14 subunit of the hetero-oligomeric complex involved in homolo...

MacromoleculeName: RPA14 subunit of the hetero-oligomeric complex involved in homologous recombination
type: protein_or_peptide / ID: 3 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Pyrococcus abyssi (archaea) / Strain: GE5 / ORSAY
Molecular weightTheoretical: 13.078994 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
RRRKPAVERK ISEIREEDTR VSLIGRVIKV DKMDYMFWLD DGTGVAIIES ESDLPKVGQV VRVIGRIIRN EEGIHIYAEV IQDFSDADL EALEEIRELE RKLLPRLEGE IVW

UniProtKB: RPA14 subunit of the hetero-oligomeric complex involved in homologous recombination

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Macromolecule #4: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.8000000000000003 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: Crystal structure published in the same study
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.35 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2) / Number images used: 141325
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 3.3.2)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 3.3.2)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 3.3.2)
FSC plot (resolution estimation)

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