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- EMDB-0725: PolD-PCNA-DNA (form B) -

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Basic information

Entry
Database: EMDB / ID: EMD-0725
TitlePolD-PCNA-DNA (form B)
Map data
Sample
  • Complex: Thermococcus kodakarensis PolD-PCNA-DNA
    • Complex: Thermococcus kodakarensis Pol D DP1 subunit
    • Complex: Thermococcus kodakarensis Pol D DP2 subunit
    • Complex: Thermococcus kodakarensis PCNA trimer ring
    • Complex: pri30DNA
    • Complex: temp45DNA
Function / homology
Function and homology information


: / : / DNA polymerase complex / exodeoxyribonuclease I / single-stranded DNA 3'-5' DNA exonuclease activity / intron homing / intein-mediated protein splicing / DNA catabolic process / DNA strand elongation involved in DNA replication / leading strand elongation ...: / : / DNA polymerase complex / exodeoxyribonuclease I / single-stranded DNA 3'-5' DNA exonuclease activity / intron homing / intein-mediated protein splicing / DNA catabolic process / DNA strand elongation involved in DNA replication / leading strand elongation / DNA polymerase processivity factor activity / regulation of DNA replication / 3'-5' exonuclease activity / DNA-templated DNA replication / endonuclease activity / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA binding / identical protein 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 / LAGLIDADG-like domain / Intein / DNA polymerase alpha/delta/epsilon, subunit B / DNA polymerase alpha/epsilon subunit B / Intein DOD homing endonuclease ...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 / LAGLIDADG-like domain / Intein / DNA polymerase alpha/delta/epsilon, subunit B / DNA polymerase alpha/epsilon subunit B / Intein DOD homing endonuclease / Intein DOD-type homing endonuclease domain profile. / Homing endonuclease, LAGLIDADG / Intein C-terminal splicing region / Intein C-terminal splicing motif profile. / Hint domain C-terminal / Hint (Hedgehog/Intein) domain C-terminal region / Intein N-terminal splicing region / Intein N-terminal splicing motif profile. / Hint domain N-terminal / Proliferating cell nuclear antigen, PCNA, conserved site / Proliferating cell nuclear antigen signature 1. / Hint (Hedgehog/Intein) domain N-terminal region / Proliferating cell nuclear antigen, PCNA / Proliferating cell nuclear antigen, PCNA, N-terminal / Proliferating cell nuclear antigen, PCNA, C-terminal / Proliferating cell nuclear antigen, N-terminal domain / Proliferating cell nuclear antigen, C-terminal domain / Homing endonuclease / Hint domain superfamily / : / Metallo-dependent phosphatase-like / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
DNA polymerase II large subunit / DNA polymerase II small subunit / DNA polymerase sliding clamp 1
Similarity search - Component
Biological speciesThermococcus kodakarensis (archaea) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.3 Å
AuthorsMayanagi K / Oki K / Miyazaki N / Ishino S / Yamagami T / Iwasaki K / Kohda D / Morikawa K / Shirai T / Ishino Y
Funding support Japan, 7 items
OrganizationGrant numberCountry
Ministry of Education, Culture, Sports, Science and Technology (Japan)18K06089 Japan
Japan Agency for Medical Research and Development (AMED)19am0101069j0003 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)17H01818 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)26242075 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)18K05442 Japan
Japan Agency for Medical Research and Development (AMED)JP19am01010720231 Japan
Japan Science and TechnologyJPMJPR12L9 Japan
CitationJournal: BMC Biol / Year: 2020
Title: Two conformations of DNA polymerase D-PCNA-DNA, an archaeal replisome complex, revealed by cryo-electron microscopy.
Authors: Kouta Mayanagi / Keisuke Oki / Naoyuki Miyazaki / Sonoko Ishino / Takeshi Yamagami / Kosuke Morikawa / Kenji Iwasaki / Daisuke Kohda / Tsuyoshi Shirai / Yoshizumi Ishino /
Abstract: BACKGROUND: DNA polymerase D (PolD) is the representative member of the D family of DNA polymerases. It is an archaea-specific DNA polymerase required for replication and unrelated to other known DNA ...BACKGROUND: DNA polymerase D (PolD) is the representative member of the D family of DNA polymerases. It is an archaea-specific DNA polymerase required for replication and unrelated to other known DNA polymerases. PolD consists of a heterodimer of two subunits, DP1 and DP2, which contain catalytic sites for 3'-5' editing exonuclease and DNA polymerase activities, respectively, with both proteins being mutually required for the full activities of each enzyme. However, the processivity of the replicase holoenzyme has additionally been shown to be enhanced by the clamp molecule proliferating cell nuclear antigen (PCNA), making it crucial to elucidate the interaction between PolD and PCNA on a structural level for a full understanding of its functional relevance. We present here the 3D structure of a PolD-PCNA-DNA complex from Thermococcus kodakarensis using single-particle cryo-electron microscopy (EM).
RESULTS: Two distinct forms of the PolD-PCNA-DNA complex were identified by 3D classification analysis. Fitting the reported crystal structures of truncated forms of DP1 and DP2 from Pyrococcus ...RESULTS: Two distinct forms of the PolD-PCNA-DNA complex were identified by 3D classification analysis. Fitting the reported crystal structures of truncated forms of DP1 and DP2 from Pyrococcus abyssi onto our EM map showed the 3D atomic structural model of PolD-PCNA-DNA. In addition to the canonical interaction between PCNA and PolD via PIP (PCNA-interacting protein)-box motif, we found a new contact point consisting of a glutamate residue at position 171 in a β-hairpin of PCNA, which mediates interactions with DP1 and DP2. The DNA synthesis activity of a mutant PolD with disruption of the E171-mediated PCNA interaction was not stimulated by PCNA in vitro.
CONCLUSIONS: Based on our analyses, we propose that glutamate residues at position 171 in each subunit of the PCNA homotrimer ring can function as hooks to lock PolD conformation on PCNA for ...CONCLUSIONS: Based on our analyses, we propose that glutamate residues at position 171 in each subunit of the PCNA homotrimer ring can function as hooks to lock PolD conformation on PCNA for conversion of its activity. This hook function of the clamp molecule may be conserved in the three domains of life.
History
DepositionJul 31, 2019-
Header (metadata) releaseAug 5, 2020-
Map releaseAug 5, 2020-
UpdateAug 5, 2020-
Current statusAug 5, 2020Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0213
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.0213
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6knc
  • Surface level: 0.0213
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0725.png

Downloads & links

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Map

FileDownload / File: emd_0725.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy AUTHOR: 0.0213 / Movie #1: 0.0213
Minimum - Maximum-0.043560315 - 0.07741171
Average (Standard dev.)0.001495657 (±0.009353208)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 176.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.11.11.1
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z176.000176.000176.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-0.0440.0770.001

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Supplemental data

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Sample components

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Entire : Thermococcus kodakarensis PolD-PCNA-DNA

EntireName: Thermococcus kodakarensis PolD-PCNA-DNA
Components
  • Complex: Thermococcus kodakarensis PolD-PCNA-DNA
    • Complex: Thermococcus kodakarensis Pol D DP1 subunit
    • Complex: Thermococcus kodakarensis Pol D DP2 subunit
    • Complex: Thermococcus kodakarensis PCNA trimer ring
    • Complex: pri30DNA
    • Complex: temp45DNA

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Supramolecule #1: Thermococcus kodakarensis PolD-PCNA-DNA

SupramoleculeName: Thermococcus kodakarensis PolD-PCNA-DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #2-#6

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Supramolecule #2: Thermococcus kodakarensis Pol D DP1 subunit

SupramoleculeName: Thermococcus kodakarensis Pol D DP1 subunit / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Thermococcus kodakarensis (archaea)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: Thermococcus kodakarensis Pol D DP2 subunit

SupramoleculeName: Thermococcus kodakarensis Pol D DP2 subunit / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Thermococcus kodakarensis (archaea)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #4: Thermococcus kodakarensis PCNA trimer ring

SupramoleculeName: Thermococcus kodakarensis PCNA trimer ring / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #4
Source (natural)Organism: Thermococcus kodakarensis (archaea)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #5: pri30DNA

SupramoleculeName: pri30DNA / type: complex / ID: 5 / Parent: 1 / Macromolecule list: #5
Details: Sequence: (DC)(DG)(DA)(DA)(DC)(DT)(DG)(DC)(DC)(DT)(DG)(DG)(DA)(DA)(DT)(DC)(DC)(DT)(DG)(DA)(DC)(DG)(DA)(DC)(DA)(DT)(DG)(DT)(DA)(DG)
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: synthetic construct (others)

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Supramolecule #6: temp45DNA

SupramoleculeName: temp45DNA / type: complex / ID: 6 / Parent: 1 / Macromolecule list: #6
Details: Sequence: (DT)(DG)(DA)(DG)(DG)(DT)(DG)(DA)(DT)(DC)(DG)(DT)(DT)(DC)(DG)(DC)(DT)(DA)(DC)(DA)(DT)(DG)(DT)(DC)(DG)(DT)(DC)(DA)(DG)(DG)(DA)(DT)(DT)(DC)(DC)(DA)(DG)(DG)(DC)(DA)(DG)(DT)(DT)(DC)(DG)
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: synthetic construct (others)

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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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Specialist opticsSpherical aberration corrector: Spherical aberration corrector
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Average electron dose: 36.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 240256
Initial angle assignmentType: COMMON LINE
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 9.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 19356
FSC plot (resolution estimation)

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