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- EMDB-50143: Pyrococcus abyssi PolD in complex with Rpa2 winged-helix domain c... -

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Basic information

Entry
Database: EMDB / ID: EMD-50143
TitlePyrococcus abyssi PolD in complex with Rpa2 winged-helix domain class 2 (composite map)
Map datadeepemhancer composite map class 2
Sample
  • Complex: PolD-Rpa2 winged-helix domain complex from Pyrococcus abyssi
    • Protein or peptide: DNA polymerase II small subunit
    • Protein or peptide: DNA polymerase II large subunit
    • Protein or peptide: RPA32 subunit of the hetero-oligomeric complex involved in homologous recombination
  • Ligand: FE (III) ION
  • Ligand: ZINC ION
KeywordsDNA Polymerase / Archaea / replication / RPA
Function / homology
Function and homology information


exodeoxyribonuclease I / DNA polymerase complex / single-stranded DNA 3'-5' DNA exonuclease activity / DNA catabolic process / DNA strand elongation involved in DNA replication / DNA-templated DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA binding
Similarity search - Function
DNA polymerase II large subunit DP2 / DNA polymerase II large subunit DP2, N-terminal / : / : / DNA polymerase II large subunit DP2, N-terminal / DNA polymerase II large subunit DP2, central domain / DNA polymerase II large subunit DP2, catalytic domain / DNA polymerase II small subunit, archaeal / : / DNA polymerase II small subunit, N-terminal domain ...DNA polymerase II large subunit DP2 / DNA polymerase II large subunit DP2, N-terminal / : / : / DNA polymerase II large subunit DP2, N-terminal / DNA polymerase II large subunit DP2, central domain / DNA polymerase II large subunit DP2, catalytic domain / DNA polymerase II small subunit, archaeal / : / DNA polymerase II small subunit, N-terminal domain / Replication factor A protein-like / DNA polymerase delta/II small subunit family / OB-fold nucleic acid binding domain, AA-tRNA synthetase-type / OB-fold nucleic acid binding domain / Calcineurin-like phosphoesterase domain, ApaH type / Calcineurin-like phosphoesterase / Metallo-dependent phosphatase-like / Winged helix-like DNA-binding domain superfamily / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
DNA polymerase II large subunit / RPA32 subunit of the hetero-oligomeric complex involved in homologous recombination / DNA polymerase II small subunit
Similarity search - Component
Biological speciesPyrococcus abyssi GE5 (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.91 Å
AuthorsMartinez-Carranza M / Sauguet L
Funding support France, European Union, 2 items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR) France
H2020 Marie Curie Actions of the European CommissionEuropean Union
CitationJournal: Nat Commun / Year: 2024
Title: Communication between DNA polymerases and Replication Protein A within the archaeal replisome.
Authors: Markel Martínez-Carranza / Léa Vialle / Clément Madru / Florence Cordier / Ayten Dizkirici Tekpinar / Ahmed Haouz / Pierre Legrand / Rémy A Le Meur / Patrick England / Rémi Dulermo / J ...Authors: Markel Martínez-Carranza / Léa Vialle / Clément Madru / Florence Cordier / Ayten Dizkirici Tekpinar / Ahmed Haouz / Pierre Legrand / Rémy A Le Meur / Patrick England / Rémi Dulermo / J Iñaki Guijarro / Ghislaine Henneke / Ludovic Sauguet /
Abstract: Replication Protein A (RPA) plays a pivotal role in DNA replication by coating and protecting exposed single-stranded DNA, and acting as a molecular hub that recruits additional replication factors. ...Replication Protein A (RPA) plays a pivotal role in DNA replication by coating and protecting exposed single-stranded DNA, and acting as a molecular hub that recruits additional replication factors. We demonstrate that archaeal RPA hosts a winged-helix domain (WH) that interacts with two key actors of the replisome: the DNA primase (PriSL) and the replicative DNA polymerase (PolD). Using an integrative structural biology approach, combining nuclear magnetic resonance, X-ray crystallography and cryo-electron microscopy, we unveil how RPA interacts with PriSL and PolD through two distinct surfaces of the WH domain: an evolutionarily conserved interface and a novel binding site. Finally, RPA is shown to stimulate the activity of PriSL in a WH-dependent manner. This study provides a molecular understanding of the WH-mediated regulatory activity in central replication factors such as RPA, which regulate genome maintenance in Archaea and Eukaryotes.
History
DepositionApr 22, 2024-
Header (metadata) releaseDec 4, 2024-
Map releaseDec 4, 2024-
UpdateJan 22, 2025-
Current statusJan 22, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50143.png

Downloads & links

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Map

FileDownload / File: emd_50143.map.gz / Format: CCP4 / Size: 163.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationdeepemhancer composite map class 2
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.96 Å/pix.
x 350 pix.
= 336. Å		0.96 Å/pix.
x 350 pix.
= 336. Å		0.96 Å/pix.
x 350 pix.
= 336. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.96 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.0017939009 - 1.832848
Average (Standard dev.)0.000832323 (±0.021144325)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions350350350
Spacing350350350
CellA=B=C: 336.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : PolD-Rpa2 winged-helix domain complex from Pyrococcus abyssi

EntireName: PolD-Rpa2 winged-helix domain complex from Pyrococcus abyssi
Components
  • Complex: PolD-Rpa2 winged-helix domain complex from Pyrococcus abyssi
    • Protein or peptide: DNA polymerase II small subunit
    • Protein or peptide: DNA polymerase II large subunit
    • Protein or peptide: RPA32 subunit of the hetero-oligomeric complex involved in homologous recombination
  • Ligand: FE (III) ION
  • Ligand: ZINC ION

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Supramolecule #1: PolD-Rpa2 winged-helix domain complex from Pyrococcus abyssi

SupramoleculeName: PolD-Rpa2 winged-helix domain complex from Pyrococcus abyssi
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Pyrococcus abyssi GE5 (archaea)
Molecular weightTheoretical: 230 KDa

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Macromolecule #1: DNA polymerase II small subunit

MacromoleculeName: DNA polymerase II small subunit / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pyrococcus abyssi GE5 (archaea)
Molecular weightTheoretical: 74.009742 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGKHHHHSGH HHTGHHHHSG SHHHTSSSAS TGENLYFQGT GDGSDELVKA LERAGYLLTP SAYYLLVDHF KEGKFSLVEL VKFAKSKGV FIIDGDLAYE FLQFLGLGVP QEIKESYIST GEEAEKTVES QETRASELEE GGVSQVSSGE LQELKEESPE I STTEEEIG ...String:
MGKHHHHSGH HHTGHHHHSG SHHHTSSSAS TGENLYFQGT GDGSDELVKA LERAGYLLTP SAYYLLVDHF KEGKFSLVEL VKFAKSKGV FIIDGDLAYE FLQFLGLGVP QEIKESYIST GEEAEKTVES QETRASELEE GGVSQVSSGE LQELKEESPE I STTEEEIG GLELVQSSIS TGSEVEYNNG ENGESVVVLD KYGYPILYAP EEIGEEKEYS KYEDVVIEWN PSVTPVQIEK NY EVKFDVR QVKLRPPKVK NGSGKEGEII VEAYASLFKS RLSKLKRILR ENPEISNVVD IGKLNYVSGD EEVTIIGLVN SKR ETNRGL IFEVEDKTGI VKVFLPKDSE DYREAFKVLP DAVVAFKGFY SKKGIFFANK FYLPDVPLYR KQKPPLEEKV YAIL ISDIH VGSREFCEKA FLKFLEWLNG HVESKEEEEI VSRVKYLIIA GDVVDGIGIY PGQYSDLVIP DIFDQYEALA NLLAN VPEH ITMFIGPGNA DAARPAIPQP EFYKEYAKPI YKLKNAIIIS NPAVIRLHGR DFLIAHGRGI EDVVSFVPGL THHKPG LPM VELLKMRHLA PTFGGKVPIA PDPEDLLVIE EVPDLVQMGH VHVYDAVVYR GVQLVNSATW QAQTEFQKMV NIVPTPA KV PVVDVESARV VKVLDFSGWC

UniProtKB: DNA polymerase II small subunit

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Macromolecule #2: DNA polymerase II large subunit

MacromoleculeName: DNA polymerase II large subunit / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Pyrococcus abyssi GE5 (archaea)
Molecular weightTheoretical: 144.418969 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MELPKEMEEY FEMLQREIDK AYEIAKKARA QGKDPSLDVE IPQATDMAGR VESLVGPPGV AKRIRELVKE YGKEIAALKI VDEIIEGKF GDLGSREKYA EQAVRTALAI LTEGIVSAPI EGIANVKIKR NTWADNSEYL ALYYAGPIRS SGGTAQALSV L VGDYVRRK ...String:
MELPKEMEEY FEMLQREIDK AYEIAKKARA QGKDPSLDVE IPQATDMAGR VESLVGPPGV AKRIRELVKE YGKEIAALKI VDEIIEGKF GDLGSREKYA EQAVRTALAI LTEGIVSAPI EGIANVKIKR NTWADNSEYL ALYYAGPIRS SGGTAQALSV L VGDYVRRK LGLDRFKPSE KHIERMVEEV DLYHRAVTRL QYHPSPEEVR LAMRNIPIEI TGEATDDVEV SHRDVPGVET NQ LRGGAIL VLAEGVLQKA KKLVKYIDKM GIEGWEWLKE FVEAKEKGEP KEEGKEESLA ESTLEETKVE VDMGFYYSLY QKF KEEIAP SDKYAKEVIG GRPLFSDPSK PGGFRLRYGR SRASGFATWG INPATMILVD EFLAIGTQLK TERPGKGAVV TPVT TIEGP IVKLKDGSVL RVDDYNLALK VREDVEEILY LGDAVIAFGD FVENNQTLLP ANYCEEWWIL EFVKALKEIY EVHLE PFTE NEEESIEEAS DYLEIDPEFL KEMLRDPLRV KPPVELAIHF SEVLGIPLHP YYTLYWNSVE PKDVEKLWRL LKNYAE IEW SNFRGIKFAK KIVISQEKLG DSKRTLELLG LPHTVRDGNV IVDYPWAAAL LTPLGNLNWE FMAKPLYATI DIINENN EI KLRDRGISWI GARMGRPEKA KERKMKPPVQ VLFPIGLAGG SSRDIKKAAE EGKVAEVEIA FFKCPKCGHV GPEHLCPN C GTRKELLWVC PRCNAEYPES QAEGYNYTCP KCNVKLRPYA KRKIRPSELL NRAMENVKVY GVDKLKGVMG MTSGWKMPE PLEKGLLRAK NDVYVFKDGT IRFDATDAPI THFRPREIGV SVEKLRELGY THDFEGKPLV SEDQIVELKP QDIILSKEAG RYLLKVAKF VDDLLEKFYG LPRFYNAEKM EDLIGHLVIG LAPHTSAGIV GRIIGFVDAL VGYAHPYFHA AKRRNCDGDE D AVMLLLDA LLNFSRYYLP EKRGGKMDAP LVITTRLDPR EVDSEVHNMD IVRYYPLEFY EATYELKSPK ELVGVIERVE DR LGKPEMY YGLKFTHDTD DIALGPKMSL YKQLGDMEEK VRRQLEVAKR IRAVDEHGVA EKILNSHLIP DLRGNLRSFT RQE FRCVKC NTKFRRPPLN GKCPVCGGKI VLTVSKGAIE KYLGTAKMLV TEYNVKNYTR QRICLTERDI DSLFENVFPE TQLT LIVNP NDICQRLVMA RTGEVNKSGL LENLSNGSKK TEKAEKAEKP RKKSDEKPKK KRVISLEEFF SRKSK

UniProtKB: DNA polymerase II large subunit

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Macromolecule #3: 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: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pyrococcus abyssi GE5 (archaea)
Molecular weightTheoretical: 15.264856 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MGKHHHHSGH HHTGHHHHSG SHHHTSSSAS TGENLYFQGT GGIMMEERSI EEPMEELLEE EIPEEKEENE LLEKAKEDIL NILRQKRTA ISRKYILKKL GDKYDEETID DAITELLAQG EIYEPETGYY KLL

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

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Macromolecule #4: FE (III) ION

MacromoleculeName: FE (III) ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: FE
Molecular weightTheoretical: 55.845 Da

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Macromolecule #5: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 5 / Number of copies: 3 / 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: FEI FALCON IV (4k 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: 2.6 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6t8h

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.91 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4) / Number images used: 174709
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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