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- EMDB-50141: Pyrococcus abyssi PolD-Rpa2 winged helix domain complex class 1 m... -

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Basic information

Entry
Database: EMDB / ID: EMD-50141
TitlePyrococcus abyssi PolD-Rpa2 winged helix domain complex class 1 main component map
Map data
Sample
  • Complex: PolD-Rpa2 winged-helix domain complex from Pyrococcus abyssi
    • Protein or peptide: PolD DP1 subunit
    • Protein or peptide: PolD DP2 subunit
    • Protein or peptide: Rpa2 winged-helix domain
KeywordsDNA Polymerase / Archaea / replication / RPA
Biological speciesPyrococcus abyssi GE5 (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 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_50141.png

Downloads & links

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Map

FileDownload / File: emd_50141.map.gz / Format: CCP4 / Size: 163.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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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)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.96 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-1.6715585 - 2.772887
Average (Standard dev.)0.00044400038 (±0.045199916)
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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Mask #1

Fileemd_50141_msk_1.map
Projections & Slices
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Half map: #2

Fileemd_50141_half_map_1.map
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Histogram

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Half map: #1

Fileemd_50141_half_map_2.map
Projections & Slices
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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: PolD DP1 subunit
    • Protein or peptide: PolD DP2 subunit
    • Protein or peptide: Rpa2 winged-helix domain

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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: all
Source (natural)Organism: Pyrococcus abyssi GE5 (archaea)
Molecular weightTheoretical: 230 KDa

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Macromolecule #1: PolD DP1 subunit

MacromoleculeName: PolD DP1 subunit / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Pyrococcus abyssi GE5 (archaea)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGKHHHHSGH HHTGHHHHSG SHHHTSSSAS TGENLYFQGT GDGSDELVKA LERAGYLLTP SAYYLLVDHF KEGKFSLVEL VKFAKSKGVF IIDGDLAYEF LQFLGLGVPQ EIKESYISTG EEAEKTVESQ ETRASELEEG GVSQVSSGEL QELKEESPEI STTEEEIGGL ...String:
MGKHHHHSGH HHTGHHHHSG SHHHTSSSAS TGENLYFQGT GDGSDELVKA LERAGYLLTP SAYYLLVDHF KEGKFSLVEL VKFAKSKGVF IIDGDLAYEF LQFLGLGVPQ EIKESYISTG EEAEKTVESQ ETRASELEEG GVSQVSSGEL QELKEESPEI STTEEEIGGL ELVQSSISTG SEVEYNNGEN GESVVVLDKY GYPILYAPEE IGEEKEYSKY EDVVIEWNPS VTPVQIEKNY EVKFDVRQVK LRPPKVKNGS GKEGEIIVEA YASLFKSRLS KLKRILRENP EISNVVDIGK LNYVSGDEEV TIIGLVNSKR ETNRGLIFEV EDKTGIVKVF LPKDSEDYRE AFKVLPDAVV AFKGFYSKKG IFFANKFYLP DVPLYRKQKP PLEEKVYAIL ISDIHVGSRE FCEKAFLKFL EWLNGHVESK EEEEIVSRVK YLIIAGDVVD GIGIYPGQYS DLVIPDIFDQ YEALANLLAN VPEHITMFIG PGNADAARPA IPQPEFYKEY AKPIYKLKNA IIISNPAVIR LHGRDFLIAH GRGIEDVVSF VPGLTHHKPG LPMVELLKMR HLAPTFGGKV PIAPDPEDLL VIEEVPDLVQ MGHVHVYDAV VYRGVQLVNS ATWQAQTEFQ KMVNIVPTPA KVPVVDVESA RVVKVLDFSG WC

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Macromolecule #2: PolD DP2 subunit

MacromoleculeName: PolD DP2 subunit / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Pyrococcus abyssi GE5 (archaea)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MELPKEMEEY FEMLQREIDK AYEIAKKARA QGKDPSLDVE IPQATDMAGR VESLVGPPGV AKRIRELVKE YGKEIAALKI VDEIIEGKFG DLGSREKYAE QAVRTALAIL TEGIVSAPIE GIANVKIKRN TWADNSEYLA LYYAGPIRSS GGTAQALSVL VGDYVRRKLG ...String:
MELPKEMEEY FEMLQREIDK AYEIAKKARA QGKDPSLDVE IPQATDMAGR VESLVGPPGV AKRIRELVKE YGKEIAALKI VDEIIEGKFG DLGSREKYAE QAVRTALAIL TEGIVSAPIE GIANVKIKRN TWADNSEYLA LYYAGPIRSS GGTAQALSVL VGDYVRRKLG LDRFKPSEKH IERMVEEVDL YHRAVTRLQY HPSPEEVRLA MRNIPIEITG EATDDVEVSH RDVPGVETNQ LRGGAILVLA EGVLQKAKKL VKYIDKMGIE GWEWLKEFVE AKEKGEPKEE GKEESLAEST LEETKVEVDM GFYYSLYQKF KEEIAPSDKY AKEVIGGRPL FSDPSKPGGF RLRYGRSRAS GFATWGINPA TMILVDEFLA IGTQLKTERP GKGAVVTPVT TIEGPIVKLK DGSVLRVDDY NLALKVREDV EEILYLGDAV IAFGDFVENN QTLLPANYCE EWWILEFVKA LKEIYEVHLE PFTENEEESI EEASDYLEID PEFLKEMLRD PLRVKPPVEL AIHFSEVLGI PLHPYYTLYW NSVEPKDVEK LWRLLKNYAE IEWSNFRGIK FAKKIVISQE KLGDSKRTLE LLGLPHTVRD GNVIVDYPWA AALLTPLGNL NWEFMAKPLY ATIDIINENN EIKLRDRGIS WIGARMGRPE KAKERKMKPP VQVLFPIGLA GGSSRDIKKA AEEGKVAEVE IAFFKCPKCG HVGPEHLCPN CGTRKELLWV CPRCNAEYPE SQAEGYNYTC PKCNVKLRPY AKRKIRPSEL LNRAMENVKV YGVDKLKGVM GMTSGWKMPE PLEKGLLRAK NDVYVFKDGT IRFDATDAPI THFRPREIGV SVEKLRELGY THDFEGKPLV SEDQIVELKP QDIILSKEAG RYLLKVAKFV DDLLEKFYGL PRFYNAEKME DLIGHLVIGL APHTSAGIVG RIIGFVDALV GYAHPYFHAA KRRNCDGDED AVMLLLDALL NFSRYYLPEK RGGKMDAPLV ITTRLDPREV DSEVHNMDIV RYYPLEFYEA TYELKSPKEL VGVIERVEDR LGKPEMYYGL KFTHDTDDIA LGPKMSLYKQ LGDMEEKVRR QLEVAKRIRA VDEHGVAEKI LNSHLIPDLR GNLRSFTRQE FRCVKCNTKF RRPPLNGKCP VCGGKIVLTV SKGAIEKYLG TAKMLVTEYN VKNYTRQRIC LTERDIDSLF ENVFPETQLT LIVNPNDICQ RLVMARTGEV NKSGLLENLS NGSKKTEKAE KAEKPRKKSD EKPKKKRVIS LEEFFSRKSK

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Macromolecule #3: Rpa2 winged-helix domain

MacromoleculeName: Rpa2 winged-helix domain / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Pyrococcus abyssi GE5 (archaea)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MGKHHHHSGH HHTGHHHHSG SHHHTSSSAS TGENLYFQGT GGIMMEERSI EEPMEELLEE EIPEEKEENE LLEKAKEDIL NILRQKRTAI SRKYILKKLG DKYDEETIDD AITELLAQGE IYEPETGYYK LL

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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: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4) / Number images used: 169361
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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