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- EMDB-16445: RPA tetrameric supercomplex with AROD-OB-1 -

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Basic information

Entry
Database: EMDB / ID: EMD-16445
TitleRPA tetrameric supercomplex with AROD-OB-1
Map data
Sample
  • Complex: RPA tetrameric supercomplex alternative conformation with AROD-OB-1
    • 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
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 (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 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 10, 2023-
Map releaseMay 10, 2023-
UpdateMay 10, 2023-
Current statusMay 10, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16445.png

Downloads & links

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Map

FileDownload / File: emd_16445.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: 0.056
Minimum - Maximum-0.48109183 - 0.7693315
Average (Standard dev.)-0.00026829494 (±0.015452279)
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_16445_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_16445_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_16445_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 alternative conformation with AROD-OB-1

EntireName: RPA tetrameric supercomplex alternative conformation with AROD-OB-1
Components
  • Complex: RPA tetrameric supercomplex alternative conformation with AROD-OB-1
    • 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 alternative conformation with AROD-OB-1

SupramoleculeName: RPA tetrameric supercomplex alternative conformation with AROD-OB-1
type: complex / ID: 1 / Chimera: Yes / 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)
Molecular weightTheoretical: 41.008965 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSVLTKDRII EIIERKTGMS REEIEEEIRK IMEEDPYLSE QGAAALLAER LGIDLIEKEE VSLMRISELY PGMDPREVNV VGRVLKKYP PREYTRKDGS VGRVASLIIY DDSGRARVVL WDAKVSEYYN KIEVGDVIKV LDAQVKESLS GLPELHINFR A RIILNPDD ...String:
MSVLTKDRII EIIERKTGMS REEIEEEIRK IMEEDPYLSE QGAAALLAER LGIDLIEKEE VSLMRISELY PGMDPREVNV VGRVLKKYP PREYTRKDGS VGRVASLIIY DDSGRARVVL WDAKVSEYYN KIEVGDVIKV LDAQVKESLS GLPELHINFR A RIILNPDD PRVEMIPPLE EVRVATYTRK KIKDIEAGDR FVEVRGTIAK VYRVLTYDAC PECKKKVDYD EGLGVWICPE HG EVQPIKM TILDFGLDDG TGYIRVTLFG DDAEELLGVS PEEIAEKIKE LEESGLTTKE AARKLAEDEF YNIIGREIVV RGN VIEDRF LGLILRASSW EDVDYRREIE RIKEELEKLG VM

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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)
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

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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)
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

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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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.8000000000000003 µm / Nominal defocus min: 0.8 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 40.0 e/Å2
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
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 3.3.2)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 3.3.2)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 3.3.2)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2) / Number images used: 41432
FSC plot (resolution estimation)

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