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- PDB-6tny: Processive human polymerase delta holoenzyme -

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Entry
Database: PDB / ID: 6tny
TitleProcessive human polymerase delta holoenzyme
Components
  • (DNA polymerase delta subunit ...) x 3
  • DNA polymerase delta catalytic subunit
  • DNA primer
  • DNA template
  • Proliferating cell nuclear antigen
KeywordsREPLICATION / Protein
Function / homology
Function and homology information


delta DNA polymerase complex / DNA synthesis involved in UV-damage excision repair / zeta DNA polymerase complex / nucleotide-excision repair complex / Cytosolic iron-sulfur cluster assembly / positive regulation of deoxyribonuclease activity / dinucleotide insertion or deletion binding / PCNA-p21 complex / mitotic telomere maintenance via semi-conservative replication / purine-specific mismatch base pair DNA N-glycosylase activity ...delta DNA polymerase complex / DNA synthesis involved in UV-damage excision repair / zeta DNA polymerase complex / nucleotide-excision repair complex / Cytosolic iron-sulfur cluster assembly / positive regulation of deoxyribonuclease activity / dinucleotide insertion or deletion binding / PCNA-p21 complex / mitotic telomere maintenance via semi-conservative replication / purine-specific mismatch base pair DNA N-glycosylase activity / 3'-5'-DNA exonuclease activity / nucleotide-excision repair, DNA gap filling / nuclear lamina / positive regulation of DNA-directed DNA polymerase activity / Polymerase switching / Telomere C-strand (Lagging Strand) Synthesis / MutLalpha complex binding / Processive synthesis on the lagging strand / DNA replication proofreading / PCNA complex / Removal of the Flap Intermediate / Processive synthesis on the C-strand of the telomere / Polymerase switching on the C-strand of the telomere / Removal of the Flap Intermediate from the C-strand / Mismatch repair (MMR) directed by MSH2:MSH3 (MutSbeta) / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / Transcription of E2F targets under negative control by DREAM complex / replisome / aggresome / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / error-free translesion synthesis / response to L-glutamate / DNA biosynthetic process / DNA synthesis involved in DNA repair / DNA strand elongation involved in DNA replication / histone acetyltransferase binding / DNA polymerase processivity factor activity / leading strand elongation / G1/S-Specific Transcription / response to dexamethasone / replication fork processing / nuclear replication fork / SUMOylation of DNA replication proteins / PCNA-Dependent Long Patch Base Excision Repair / fatty acid homeostasis / error-prone translesion synthesis / translesion synthesis / mismatch repair / response to cadmium ion / response to UV / estrous cycle / cyclin-dependent protein kinase holoenzyme complex / base-excision repair, gap-filling / DNA polymerase binding / positive regulation of endothelial cell proliferation / epithelial cell differentiation / male germ cell nucleus / positive regulation of DNA repair / TP53 Regulates Transcription of Genes Involved in G2 Cell Cycle Arrest / Translesion synthesis by REV1 / Translesion synthesis by POLK / liver regeneration / Translesion synthesis by POLI / replication fork / Gap-filling DNA repair synthesis and ligation in GG-NER / positive regulation of DNA replication / nuclear estrogen receptor binding / Termination of translesion DNA synthesis / Recognition of DNA damage by PCNA-containing replication complex / Translesion Synthesis by POLH / receptor tyrosine kinase binding / HDR through Homologous Recombination (HRR) / Dual Incision in GG-NER / cellular response to hydrogen peroxide / DNA-templated DNA replication / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / cellular response to UV / cellular response to xenobiotic stimulus / E3 ubiquitin ligases ubiquitinate target proteins / response to estradiol / heart development / 4 iron, 4 sulfur cluster binding / protein-macromolecule adaptor activity / damaged DNA binding / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / chromosome, telomeric region / DNA replication / nuclear body / DNA repair / nucleotide binding / centrosome / chromatin binding / protein-containing complex binding / chromatin / enzyme binding / negative regulation of transcription by RNA polymerase II / DNA binding / extracellular exosome
Similarity search - Function
DNA polymerase delta, p66 (Cdc27) subunit, wHTH domain / OB fold (Dihydrolipoamide Acetyltransferase, E2P) - #430 / Purple Acid Phosphatase; chain A, domain 2 - #50 / DNA polymerase delta, subunit 4 / DNA polymerase delta, subunit 4 / DNA polymerase delta subunit 3 / DNA polymerase delta subunit 3 superfamily / DNA polymerase subunit Cdc27 / DNA polymerase delta subunit, OB-fold domain / DNA polymerase delta subunit 2, C-terminal domain ...DNA polymerase delta, p66 (Cdc27) subunit, wHTH domain / OB fold (Dihydrolipoamide Acetyltransferase, E2P) - #430 / Purple Acid Phosphatase; chain A, domain 2 - #50 / DNA polymerase delta, subunit 4 / DNA polymerase delta, subunit 4 / DNA polymerase delta subunit 3 / DNA polymerase delta subunit 3 superfamily / DNA polymerase subunit Cdc27 / DNA polymerase delta subunit, OB-fold domain / DNA polymerase delta subunit 2, C-terminal domain / DNA polymerase delta subunit OB-fold domain / DNA polymerase delta/II small subunit family / C4-type zinc-finger of DNA polymerase delta / : / C4-type zinc-finger of DNA polymerase delta / DNA polymerase delta catalytic subunit-like, N-terminal domain / 50s Ribosomal Protein L17; Chain: A, / Box / Proliferating Cell Nuclear Antigen / Proliferating Cell Nuclear Antigen - #10 / DNA polymerase alpha/delta/epsilon, subunit B / DNA polymerase alpha/epsilon subunit B / Proliferating cell nuclear antigen signature 2. / : / Proliferating cell nuclear antigen, PCNA, conserved site / Proliferating cell nuclear antigen signature 1. / 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 / Purple Acid Phosphatase; chain A, domain 2 / DNA polymerase family B, thumb domain / DNA polymerase family B signature. / DNA-directed DNA polymerase, family B, multifunctional domain / DNA-directed DNA polymerase, family B, conserved site / DNA polymerase family B / : / DNA polymerase family B, exonuclease domain / DNA-directed DNA polymerase, family B, exonuclease domain / DNA polymerase, palm domain superfamily / DNA polymerase type-B family / DNA-directed DNA polymerase, family B / 4-Layer Sandwich / OB fold (Dihydrolipoamide Acetyltransferase, E2P) / Ribonuclease H superfamily / Ribonuclease H-like superfamily / DNA/RNA polymerase superfamily / Alpha-Beta Complex / Beta Barrel / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
IRON/SULFUR CLUSTER / THYMIDINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / Proliferating cell nuclear antigen / DNA polymerase delta catalytic subunit / DNA polymerase delta subunit 2 / DNA polymerase delta subunit 3 / DNA polymerase delta subunit 4
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.08 Å
AuthorsLancey, C. / Hamdan, S.M. / De Biasio, A.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust United Kingdom
CitationJournal: Nat Commun / Year: 2020
Title: Structure of the processive human Pol δ holoenzyme.
Authors: Claudia Lancey / Muhammad Tehseen / Vlad-Stefan Raducanu / Fahad Rashid / Nekane Merino / Timothy J Ragan / Christos G Savva / Manal S Zaher / Afnan Shirbini / Francisco J Blanco / Samir M ...Authors: Claudia Lancey / Muhammad Tehseen / Vlad-Stefan Raducanu / Fahad Rashid / Nekane Merino / Timothy J Ragan / Christos G Savva / Manal S Zaher / Afnan Shirbini / Francisco J Blanco / Samir M Hamdan / Alfredo De Biasio /
Abstract: In eukaryotes, DNA polymerase δ (Pol δ) bound to the proliferating cell nuclear antigen (PCNA) replicates the lagging strand and cooperates with flap endonuclease 1 (FEN1) to process the Okazaki ...In eukaryotes, DNA polymerase δ (Pol δ) bound to the proliferating cell nuclear antigen (PCNA) replicates the lagging strand and cooperates with flap endonuclease 1 (FEN1) to process the Okazaki fragments for their ligation. We present the high-resolution cryo-EM structure of the human processive Pol δ-DNA-PCNA complex in the absence and presence of FEN1. Pol δ is anchored to one of the three PCNA monomers through the C-terminal domain of the catalytic subunit. The catalytic core sits on top of PCNA in an open configuration while the regulatory subunits project laterally. This arrangement allows PCNA to thread and stabilize the DNA exiting the catalytic cleft and recruit FEN1 to one unoccupied monomer in a toolbelt fashion. Alternative holoenzyme conformations reveal important functional interactions that maintain PCNA orientation during synthesis. This work sheds light on the structural basis of Pol δ's activity in replicating the human genome.
History
DepositionDec 10, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 18, 2019Provider: repository / Type: Initial release
Revision 1.1Mar 11, 2020Group: 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 / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2May 22, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / struct_conn / struct_conn_type
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.conn_type_id / _struct_conn.id / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id / _struct_conn_type.id

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: DNA polymerase delta catalytic subunit
B: DNA polymerase delta subunit 2
C: DNA polymerase delta subunit 3
D: DNA polymerase delta subunit 4
E: Proliferating cell nuclear antigen
F: Proliferating cell nuclear antigen
G: Proliferating cell nuclear antigen
P: DNA primer
T: DNA template
hetero molecules


Theoretical massNumber of molelcules
Total (without water)351,43412
Polymers350,5359
Non-polymers8993
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area25370 Å2
ΔGint-133 kcal/mol
Surface area114650 Å2
MethodPISA

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Components

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Protein , 2 types, 4 molecules AEFG

#1: Protein DNA polymerase delta catalytic subunit / DNA polymerase subunit delta p125


Mass: 123785.922 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: POLD1, POLD / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P28340, DNA-directed DNA polymerase, Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters
#5: Protein Proliferating cell nuclear antigen / PCNA / Cyclin


Mass: 29088.061 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PCNA / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P12004

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DNA polymerase delta subunit ... , 3 types, 3 molecules BCD

#2: Protein DNA polymerase delta subunit 2 / DNA polymerase delta subunit p50


Mass: 51338.168 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: POLD2 / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P49005
#3: Protein DNA polymerase delta subunit 3 / DNA polymerase delta subunit C / DNA polymerase delta subunit p66 / DNA polymerase delta subunit p68


Mass: 52528.500 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: POLD3, KIAA0039 / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q15054
#4: Protein DNA polymerase delta subunit 4 / DNA polymerase delta subunit p12


Mass: 16274.323 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: POLD4, POLDS / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9HCU8

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DNA chain , 2 types, 2 molecules PT

#6: DNA chain DNA primer


Mass: 7665.987 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#7: DNA chain DNA template


Mass: 11677.539 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 3 types, 3 molecules

#8: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#9: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe4S4
#10: Chemical ChemComp-TTP / THYMIDINE-5'-TRIPHOSPHATE


Mass: 482.168 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N2O14P3

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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

Component
IDNameTypeEntity IDParent-IDSource
1ToolbeltCOMPLEX#1-#5, #70MULTIPLE SOURCES
2DNA polymeraseCOMPLEX#1-#41RECOMBINANT
3Proliferating cell nuclear antigenCOMPLEX#51RECOMBINANT
4DNA primer, templateCOMPLEX#6-#71RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Homo sapiens (human)9606
34synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Spodoptera frugiperda (fall armyworm)7108
23Escherichia coli BL21(DE3) (bacteria)469008
34synthetic construct (others)32630
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHEPESC8H18N2O4S1
2100 mMPotassium AcetateCH3CO2K1
310 mMCalcium ChlorideCaCl21
40.02 %NP-40H(C2H4O)nO(C6H4)C9H191
50.4 mMBiotinC10H16N2O3S1
61 mMDithiothreitolC4H10O2S21
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Complex separated by gel filtration
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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 FIELD / Nominal magnification: 105000 X / Calibrated magnification: 57471 X / Nominal defocus max: 2300 nm / Nominal defocus min: 1100 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 77 K / Temperature (min): 77 K
Image recordingAverage exposure time: 3 sec. / Electron dose: 44 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5071 / Details: Data were collected in super resolution mode
Image scansWidth: 11520 / Height: 8184

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameVersionCategory
2EPU2.4.0.54image acquisition
4CTFFIND4.1CTF correction
11RELION3.1final Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.08 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 288920 / Symmetry type: POINT

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