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- PDB-6hms: Cryo-EM map of DNA polymerase D from Pyrococcus abyssi in complex... -

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Basic information

Entry
Database: PDB / ID: 6hms
TitleCryo-EM map of DNA polymerase D from Pyrococcus abyssi in complex with DNA
Components
  • (DNA polymerase II ...) x 2
  • DNA (5'-D(*GP*AP*GP*AP*CP*GP*GP*GP*CP*CP*GP*CP*GP*TP*C)-3')
  • DNA (5'-D(P*TP*GP*AP*CP*GP*CP*GP*GP*CP*CP*CP*GP*TP*CP*TP*C)-3')
KeywordsREPLICATION / DNA / polymerase D / Pyrococcus
Function / homology
Function and homology information


exodeoxyribonuclease I / single-stranded DNA 3'-5' DNA exonuclease activity / intein-mediated protein splicing / DNA catabolic process / 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 / DNA polymerase II small subunit, archaeal / DNA polymerase delta/II small subunit family / Intein splicing domain / Intein C-terminal splicing region / Intein C-terminal splicing motif profile. / Intein N-terminal splicing region / Intein N-terminal splicing motif profile. ...DNA polymerase II large subunit DP2 / DNA polymerase II large subunit DP2, N-terminal / DNA polymerase II large subunit DP2 / DNA polymerase II small subunit, archaeal / DNA polymerase delta/II small subunit family / Intein splicing domain / Intein C-terminal splicing region / Intein C-terminal splicing motif profile. / Intein N-terminal splicing region / Intein N-terminal splicing motif profile. / Hint domain N-terminal / Hint (Hedgehog/Intein) domain N-terminal region / Hint domain superfamily / Calcineurin-like phosphoesterase domain, ApaH type / Calcineurin-like phosphoesterase / Metallo-dependent phosphatase-like / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
: / DNA / DNA (> 10) / DNA polymerase II small subunit / DNA polymerase II large subunit
Similarity search - Component
Biological speciesPyrococcus abyssi (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.1 Å
AuthorsRaia, P. / Carroni, M. / Sauguet, L.
Funding support France, 1items
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.
History
DepositionSep 12, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 30, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 11, 2019Group: Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB

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Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-0244
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: DNA polymerase II small subunit
B: DNA polymerase II large subunit,DNA polymerase II large subunit
P: DNA (5'-D(*GP*AP*GP*AP*CP*GP*GP*GP*CP*CP*GP*CP*GP*TP*C)-3')
T: DNA (5'-D(P*TP*GP*AP*CP*GP*CP*GP*GP*CP*CP*CP*GP*TP*CP*TP*C)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)199,3029
Polymers198,9844
Non-polymers3175
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA polymerase II ... , 2 types, 2 molecules AB

#1: Protein DNA polymerase II small subunit / / Pol II / Exodeoxyribonuclease small subunit / Coordinate model: Cα atoms only


Mass: 45078.012 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus abyssi (archaea) / Gene: polB, PYRAB01210, PAB2266 / Production host: Escherichia coli (E. coli)
References: UniProt: Q9V2F3, DNA-directed DNA polymerase, exodeoxyribonuclease I
#2: Protein DNA polymerase II large subunit,DNA polymerase II large subunit / / Pol II / Exodeoxyribonuclease large subunit / Coordinate model: Cα atoms only


Mass: 144418.969 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus abyssi (archaea) / Gene: polC, PYRAB01200, PAB2404 / Production host: Escherichia coli (E. coli)
References: UniProt: Q9V2F4, DNA-directed DNA polymerase, exodeoxyribonuclease I

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DNA chain , 2 types, 2 molecules PT

#3: DNA chain DNA (5'-D(*GP*AP*GP*AP*CP*GP*GP*GP*CP*CP*GP*CP*GP*TP*C)-3')


Mass: 4635.998 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Pyrococcus abyssi (archaea)
#4: DNA chain DNA (5'-D(P*TP*GP*AP*CP*GP*CP*GP*GP*CP*CP*CP*GP*TP*CP*TP*C)-3')


Mass: 4851.129 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Pyrococcus abyssi (archaea)

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Non-polymers , 2 types, 5 molecules

#5: Chemical ChemComp-FE / FE (III) ION / Iron


Mass: 55.845 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe
#6: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Complex between DNA polymerase D and DNA duplexCOMPLEX#1-#40MULTIPLE SOURCES
2DNA polymerase DCOMPLEX#1-#21RECOMBINANT
3DNACOMPLEX#3-#41RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Pyrococcus abyssi (archaea)29292
33Pyrococcus abyssi (archaea)29292
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33synthetic construct (others)32630
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 1 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM softwareName: cryoSPARC / Category: 3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 7.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 8774 / Symmetry type: POINT
RefinementHighest resolution: 7.1 Å
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00812947
ELECTRON MICROSCOPYf_angle_d1.37417637
ELECTRON MICROSCOPYf_dihedral_angle_d9.9647746
ELECTRON MICROSCOPYf_chiral_restr0.0731940
ELECTRON MICROSCOPYf_plane_restr0.012164

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