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- PDB-6knc: PolD-PCNA-DNA (form B) -

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Basic information

Entry
Database: PDB / ID: 6knc
TitlePolD-PCNA-DNA (form B)
Components
  • (DNA polymerase ...) x 3
  • pri30DNA
  • temp45DNA
KeywordsREPLICATION/DNA / Family D Polymerase / PCNA / Switch Hook / REPLICATION / REPLICATION-DNA complex
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 / DNA (> 10) / DNA polymerase II large subunit / DNA polymerase II small subunit / DNA polymerase sliding clamp 1
Similarity search - Component
Biological speciesThermococcus kodakarensis KOD1 (archaea)
Thermococcus kodakarensis (archaea)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single 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, 7items
OrganizationGrant numberCountry
Ministry of Education, Culture, Sports, Science and Technology (Japan)18K06089 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)17H01818 Japan
Japan Agency for Medical Research and Development (AMED)20am0101069j0004 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)18K05442 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)26242075 Japan
Japan Science and TechnologyJPMJPR12L9 Japan
Japan Agency for Medical Research and Development (AMED)JP19am01010720231 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
DepositionAug 5, 2019Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 5, 2020Provider: repository / Type: Initial release
Revision 1.1Feb 17, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Mar 27, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: DNA polymerase II small subunit
B: DNA polymerase D DP2 (DNA polymerase II large) subunit
C: DNA polymerase sliding clamp 1
D: DNA polymerase sliding clamp 1
E: DNA polymerase sliding clamp 1
F: pri30DNA
G: temp45DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)314,76712
Polymers314,4507
Non-polymers3175
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area15170 Å2
ΔGint-127 kcal/mol
Surface area116150 Å2

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Components

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DNA polymerase ... , 3 types, 5 molecules ABCDE

#1: Protein DNA polymerase II small subunit / / DNA polymerase D DP1 (DNA polymerase II small) subunit / Pol II / Exodeoxyribonuclease small subunit


Mass: 60772.336 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermococcus kodakarensis KOD1 (archaea)
Strain: ATCC BAA-918 / JCM 12380 / KOD1 / Gene: polB, TK1902 / Production host: Escherichia coli (E. coli)
References: UniProt: Q5JET1, DNA-directed DNA polymerase, exodeoxyribonuclease I
#2: Protein DNA polymerase D DP2 (DNA polymerase II large) subunit


Mass: 150418.047 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermococcus kodakarensis (archaea) / Production host: Escherichia coli (E. coli) / References: UniProt: Q5JET0*PLUS
#3: Protein DNA polymerase sliding clamp 1 / DNA clamp / Proliferating cell nuclear antigen homolog 1 / PCNA 1


Mass: 28270.441 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermococcus kodakarensis KOD1 (archaea)
Strain: ATCC BAA-918 / JCM 12380 / KOD1 / Gene: pcn1, TK0535 / Production host: Escherichia coli (E. coli) / References: UniProt: Q5JF32

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DNA chain , 2 types, 2 molecules FG

#4: DNA chain pri30DNA


Mass: 9232.954 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: DNA chain temp45DNA


Mass: 9214.928 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 2 types, 5 molecules

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


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


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

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Details

Has ligand of interestN

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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

ComponentName: Thermococcus kodakarensis PolD-PCNA-DNA / Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Molecular weightValue: 0.340 MDa / Experimental value: NO
Source (natural)Organism: Thermococcus kodakarensis (archaea)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: Quantifoil R1.2/1.3
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: 36 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON II (4k x 4k)
EM imaging opticsPhase plate: VOLTA PHASE PLATE
Spherical aberration corrector: Spherical aberration corrector

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Processing

SoftwareName: PHENIX / Version: (1.11.1_2575: phenix.real_space_refine) / Classification: refinement
EM software
IDNameCategory
7UCSF Chimeramodel fitting
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 240256
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 9.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 19356 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model building

3D fitting-ID: 1 / Pdb chain-ID: A / Source name: PDB / Type: experimental model

IDPDB-IDAccession codeInitial refinement model-ID
15IHE

5ihe

5IHE1
25IJL

5ijl

5IJL2
33LX1

3lx1

3LX13
RefinementStereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 438.08 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.009820037
ELECTRON MICROSCOPYf_angle_d0.988827268
ELECTRON MICROSCOPYf_chiral_restr0.05663037
ELECTRON MICROSCOPYf_plane_restr0.00633357
ELECTRON MICROSCOPYf_dihedral_angle_d12.98257735

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