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- EMDB-0244: Cryo-EM map of DNA polymerase D from Pyrococcus abyssi in complex... -

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Basic information

Entry
Database: EMDB / ID: EMD-0244
TitleCryo-EM map of DNA polymerase D from Pyrococcus abyssi in complex with DNA
Map data
SampleComplex between DNA polymerase D and DNA duplex:
DNA polymerase D / DNA / (DNA polymerase II ...) x 2 / (nucleic-acidNucleic acid) x 2 / (ligand) x 2
Function / homology
Function and homology information


exodeoxyribonuclease I activity / exodeoxyribonuclease I / DNA catabolic process, exonucleolytic / intein-mediated protein splicing / 3'-5' exonuclease activity / DNA-dependent DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA binding
DNA polymerase II large subunit DP2 / DNA polymerase delta/II small subunit family / Hint domain superfamily / Intein C-terminal splicing region / Hint domain N-terminal / Calcineurin-like phosphoesterase domain, ApaH type / Intein N-terminal splicing region / DNA polymerase II small subunit, archaeal / Nucleic acid-binding, OB-fold / DNA polymerase II large subunit DP2, N-terminal
DNA polymerase II small subunit / DNA polymerase II large subunit
Biological speciesPyrococcus abyssi (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.1 Å
AuthorsRaia P / Carroni M / Sauguet L
Funding support France, 1 items
OrganizationGrant numberCountry
French National Research AgencyANR -17-CE11-0005 France
CitationJournal: PLoS Biol. / Year: 2019
Title: Structure of the DP1-DP2 PolD complex bound with DNA and its implications for the evolutionary history of DNA and RNA polymerases.
Authors: Pierre Raia / Marta Carroni / Etienne Henry / Gérard Pehau-Arnaudet / Sébastien Brûlé / Pierre Béguin / Ghislaine Henneke / Erik Lindahl / Marc Delarue / Ludovic Sauguet /
Abstract: PolD is an archaeal replicative DNA polymerase (DNAP) made of a proofreading exonuclease subunit (DP1) and a larger polymerase catalytic subunit (DP2). Recently, we reported the individual crystal ...PolD is an archaeal replicative DNA polymerase (DNAP) made of a proofreading exonuclease subunit (DP1) and a larger polymerase catalytic subunit (DP2). Recently, we reported the individual crystal structures of the DP1 and DP2 catalytic cores, thereby revealing that PolD is an atypical DNAP that has all functional properties of a replicative DNAP but with the catalytic core of an RNA polymerase (RNAP). We now report the DNA-bound cryo-electron microscopy (cryo-EM) structure of the heterodimeric DP1-DP2 PolD complex from Pyrococcus abyssi, revealing a unique DNA-binding site. Comparison of PolD and RNAPs extends their structural similarities and brings to light the minimal catalytic core shared by all cellular transcriptases. Finally, elucidating the structure of the PolD DP1-DP2 interface, which is conserved in all eukaryotic replicative DNAPs, clarifies their evolutionary relationships with PolD and sheds light on the domain acquisition and exchange mechanism that occurred during the evolution of the eukaryotic replisome.
Validation ReportPDB-ID: 6hms

SummaryFull reportAbout validation report
History
DepositionSep 12, 2018-
Header (metadata) releaseOct 10, 2018-
Map releaseJan 30, 2019-
UpdateDec 11, 2019-
Current statusDec 11, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.58
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.58
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: : PDB-6hms
  • Surface level: 0.58
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_0244.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.36 Å/pix.
x 220 pix.
= 299.2 Å
1.36 Å/pix.
x 220 pix.
= 299.2 Å
1.36 Å/pix.
x 220 pix.
= 299.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.36 Å
Density
Contour LevelBy AUTHOR: 0.58 / Movie #1: 0.58
Minimum - Maximum-3.568817 - 3.8639772
Average (Standard dev.)0.0047397213 (±0.42160863)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 299.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.361.361.36
M x/y/z220220220
origin x/y/z0.0000.0000.000
length x/y/z299.200299.200299.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS220220220
D min/max/mean-0.6581.6240.005

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Supplemental data

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Half map: cryo-EM half map A of DNA polymerase D...

Fileemd_0244_half_map_1.map
Annotationcryo-EM half map A of DNA polymerase D from Pyrococcus abyssi in complex with DNA
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: cryo-EM half map B of DNA polymerase D...

Fileemd_0244_half_map_2.map
Annotationcryo-EM half map B of DNA polymerase D from Pyrococcus abyssi in complex with DNA
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire Complex between DNA polymerase D and DNA duplex

EntireName: Complex between DNA polymerase D and DNA duplex / Number of components: 9

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Component #1: protein, Complex between DNA polymerase D and DNA duplex

ProteinName: Complex between DNA polymerase D and DNA duplex / Recombinant expression: No

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Component #2: protein, DNA polymerase D

ProteinName: DNA polymerase D / Recombinant expression: No
SourceSpecies: Pyrococcus abyssi (archaea)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #3: protein, DNA

ProteinName: DNA / Recombinant expression: No
SourceSpecies: Pyrococcus abyssi (archaea)
Source (engineered)Expression System: synthetic construct (others)

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Component #4: protein, DNA polymerase II small subunit

ProteinName: DNA polymerase II small subunit / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 45.078012 kDa
SourceSpecies: Pyrococcus abyssi (archaea)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #5: protein, DNA polymerase II large subunit,DNA polymerase II large ...

ProteinName: DNA polymerase II large subunit,DNA polymerase II large subunit
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 144.418969 kDa
SourceSpecies: Pyrococcus abyssi (archaea)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #6: nucleic-acid, DNA (5'-D(*GP*AP*GP*AP*CP*GP*GP*GP*CP*CP*GP*CP*GP*T...

nucleic acidName: DNA (5'-D(*GP*AP*GP*AP*CP*GP*GP*GP*CP*CP*GP*CP*GP*TP*C)-3')
Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DG)(DA)(DG)(DA)(DC)(DG)(DG)(DG)(DC)(DC) (DG)(DC)(DG)(DT)(DC)
MassTheoretical: 4.635998 kDa
SourceSpecies: Pyrococcus abyssi (archaea)

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Component #7: nucleic-acid, DNA (5'-D(P*TP*GP*AP*CP*GP*CP*GP*GP*CP*CP*CP*GP*TP*...

nucleic acidName: DNA (5'-D(P*TP*GP*AP*CP*GP*CP*GP*GP*CP*CP*CP*GP*TP*CP*TP*C)-3')
Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DT)(DG)(DA)(DC)(DG)(DC)(DG)(DG)(DC)(DC) (DC)(DG)(DT)(DC)(DT)(DC)
MassTheoretical: 4.851129 kDa
SourceSpecies: Pyrococcus abyssi (archaea)

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Component #8: ligand, FE (III) ION

LigandName: FE (III) ION / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 5.584505 MDa

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Component #9: ligand, ZINC ION

LigandName: ZINC ION / Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 6.540905 MDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 QUANTUM (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 8774
3D reconstructionSoftware: cryoSPARC / Resolution: 7.1 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Output model

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