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- EMDB-16448: Replication Protein A bound to DNA -

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Basic information

Entry
Database: EMDB / ID: EMD-16448
TitleReplication Protein A bound to DNA
Map data
Sample
  • Complex: RPA bound to a 100-mer poly-dT ssDNA
    • Complex: RPA 1, 2 and 3
      • Protein or peptide: RPA1
      • Protein or peptide: RPA2
      • Protein or peptide: RPA3
    • Complex: poly dT
      • DNA: poly dT
Biological speciesPyrococcus abyssi (archaea) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 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-
UpdateMay 3, 2023-
Current statusMay 3, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16448.png

Downloads & links

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Map

FileDownload / File: emd_16448.map.gz / Format: CCP4 / Size: 347.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.76 Å/pix.
x 450 pix.
= 342. Å		0.76 Å/pix.
x 450 pix.
= 342. Å		0.76 Å/pix.
x 450 pix.
= 342. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.76 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.12
Minimum - Maximum-1.0532295 - 1.3625758
Average (Standard dev.)-7.964999e-05 (±0.010684242)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions450450450
Spacing450450450
CellA=B=C: 342.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16448_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_16448_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_16448_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 bound to a 100-mer poly-dT ssDNA

EntireName: RPA bound to a 100-mer poly-dT ssDNA
Components
  • Complex: RPA bound to a 100-mer poly-dT ssDNA
    • Complex: RPA 1, 2 and 3
      • Protein or peptide: RPA1
      • Protein or peptide: RPA2
      • Protein or peptide: RPA3
    • Complex: poly dT
      • DNA: poly dT

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Supramolecule #1: RPA bound to a 100-mer poly-dT ssDNA

SupramoleculeName: RPA bound to a 100-mer poly-dT ssDNA / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 180 KDa

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Supramolecule #2: RPA 1, 2 and 3

SupramoleculeName: RPA 1, 2 and 3 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#3
Source (natural)Organism: Pyrococcus abyssi (archaea)

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Supramolecule #3: poly dT

SupramoleculeName: poly dT / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #4
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: RPA1

MacromoleculeName: RPA1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Pyrococcus abyssi (archaea)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSVLTKDRII EIIERKTGMS REEIEEEIRK IMEEDPYLSE QGAAALLAER LGIDLIEKEE VSLMRISELY PGMDPREVNV VGRVLKKYPP REYTRKDGSV GRVASLIIYD DSGRARVVLW DAKVSEYYNK IEVGDVIKVL DAQVKESLSG LPELHINFRA RIILNPDDPR ...String:
MSVLTKDRII EIIERKTGMS REEIEEEIRK IMEEDPYLSE QGAAALLAER LGIDLIEKEE VSLMRISELY PGMDPREVNV VGRVLKKYPP REYTRKDGSV GRVASLIIYD DSGRARVVLW DAKVSEYYNK IEVGDVIKVL DAQVKESLSG LPELHINFRA RIILNPDDPR VEMIPPLEEV RVATYTRKKI KDIEAGDRFV EVRGTIAKVY RVLTYDACPE CKKKVDYDEG LGVWICPEHG EVQPIKMTIL DFGLDDGTGY IRVTLFGDDA EELLGVSPEE IAEKIKELEE SGLTTKEAAR KLAEDEFYNI IGREIVVRGN VIEDRFLGLI LRASSWEDVD YRREIERIKE ELEKLGVM

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Macromolecule #2: RPA2

MacromoleculeName: RPA2 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Pyrococcus abyssi (archaea)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSKKRMPATR LYIKDILEGY FVKSEGDFEP NYLITKYARK VYRAKIVGTV VREPLIAEDE TYGKFQVDDG TGVIWVLGFR DDTKFAKLVR KGDLVQVIGK IAEWRDDKQI LVEGVSKVHP NMWILHRYET LKEKIEHIKK AKIALEIYNQ YGITAKSKVI AKNKGIEEEL ...String:
MSKKRMPATR LYIKDILEGY FVKSEGDFEP NYLITKYARK VYRAKIVGTV VREPLIAEDE TYGKFQVDDG TGVIWVLGFR DDTKFAKLVR KGDLVQVIGK IAEWRDDKQI LVEGVSKVHP NMWILHRYET LKEKIEHIKK AKIALEIYNQ YGITAKSKVI AKNKGIEEEL LEVIDELYGI MMEERSIEEP MEELLEEEIP EEKEENELLE KAKEDILNIL RQKRTAISRK YILKKLGDKY DEETIDDAIT ELLAQGEIYE PETGYYKLL

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Macromolecule #3: RPA3

MacromoleculeName: RPA3 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Pyrococcus abyssi (archaea)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
GTGDGSEVQV RRRKPAVERK ISEIREEDTR VSLIGRVIKV DKMDYMFWLD DGTGVAIIES ESDLPKVGQV VRVIGRIIRN EEGIHIYAEV IQDFSDADLE ALEEIRELER KLLPRLEGEI VW

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Macromolecule #4: poly dT

MacromoleculeName: poly dT / type: dna / ID: 4 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
SequenceString:
TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT

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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

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Detector mode: INTEGRATING / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 200 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

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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: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2) / Number images used: 126856
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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