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- EMDB-30937: PolD-primase -

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Basic information

Entry
Database: EMDB / ID: EMD-30937
TitlePolD-primase
Map data
Sample
  • Complex: PolD-primase
Biological speciesThermococcus kodakarensis KOD1 (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.1 Å
AuthorsOki K / Mayanagi K / Ishino Y
Funding support Japan, 9 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)JP20J12260 Japan
Japan Society for the Promotion of Science (JSPS)JP26242075 Japan
Japan Society for the Promotion of Science (JSPS)JP18K05442 Japan
Japan Society for the Promotion of Science (JSPS)JP18K06089 Japan
Japan Society for the Promotion of Science (JSPS)JP16H01410 Japan
Japan Society for the Promotion of Science (JSPS)JP17H01818 Japan
Japan Science and TechnologyJPMJPR12L9 Japan
Japan Agency for Medical Research and Development (AMED)JP19am0101069 Japan
Japan Agency for Medical Research and Development (AMED)JP21am0101071 Japan
CitationJournal: Nucleic Acids Res / Year: 2021
Title: DNA polymerase D temporarily connects primase to the CMG-like helicase before interacting with proliferating cell nuclear antigen.
Authors: Keisuke Oki / Takeshi Yamagami / Mariko Nagata / Kouta Mayanagi / Tsuyoshi Shirai / Naruhiko Adachi / Tomoyuki Numata / Sonoko Ishino / Yoshizumi Ishino /
Abstract: The eukaryotic replisome is comprised of three family-B DNA polymerases (Polα, δ and ϵ). Polα forms a stable complex with primase to synthesize short RNA-DNA primers, which are subsequently ...The eukaryotic replisome is comprised of three family-B DNA polymerases (Polα, δ and ϵ). Polα forms a stable complex with primase to synthesize short RNA-DNA primers, which are subsequently elongated by Polδ and Polϵ in concert with proliferating cell nuclear antigen (PCNA). In some species of archaea, family-D DNA polymerase (PolD) is the only DNA polymerase essential for cell viability, raising the question of how it alone conducts the bulk of DNA synthesis. We used a hyperthermophilic archaeon, Thermococcus kodakarensis, to demonstrate that PolD connects primase to the archaeal replisome before interacting with PCNA. Whereas PolD stably connects primase to GINS, a component of CMG helicase, cryo-EM analysis indicated a highly flexible PolD-primase complex. A conserved hydrophobic motif at the C-terminus of the DP2 subunit of PolD, a PIP (PCNA-Interacting Peptide) motif, was critical for the interaction with primase. The dissociation of primase was induced by DNA-dependent binding of PCNA to PolD. Point mutations in the alternative PIP-motif of DP2 abrogated the molecular switching that converts the archaeal replicase from de novo to processive synthesis mode.
History
DepositionJan 30, 2021-
Header (metadata) releaseApr 28, 2021-
Map releaseApr 28, 2021-
UpdateMay 19, 2021-
Current statusMay 19, 2021Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.00664
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.00664
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30937.png

Downloads & links

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Map

FileDownload / File: emd_30937.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.86 Å
Density
Contour LevelBy AUTHOR: 0.00664 / Movie #1: 0.00664
Minimum - Maximum-0.023137338 - 0.04849132
Average (Standard dev.)0.00019965094 (±0.0019223987)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 275.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.860.860.86
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z275.200275.200275.200
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ512512512
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0230.0480.000

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Supplemental data

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Sample components

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Entire : PolD-primase

EntireName: PolD-primase
Components
  • Complex: PolD-primase

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Supramolecule #1: PolD-primase

SupramoleculeName: PolD-primase / type: complex / ID: 1 / Parent: 0 / Details: PolD-primase complex
Source (natural)Organism: Thermococcus kodakarensis KOD1 (archaea)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)

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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
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Average electron dose: 49.92 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 7.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 85888
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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