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- PDB-6s1n: Human polymerase delta holoenzyme Conformer 2 -

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Basic information

Entry
Database: PDB / ID: 6s1n
TitleHuman polymerase delta holoenzyme Conformer 2
Components
  • (DNA polymerase delta subunit ...) x 3
  • DNA polymerase delta catalytic subunit
  • DNA primerPrimer (molecular biology)
  • 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 / positive regulation of deoxyribonuclease activity / dinucleotide insertion or deletion binding / PCNA-p21 complex / mitotic telomere maintenance via semi-conservative replication / Cytosolic iron-sulfur cluster assembly / 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 / positive regulation of deoxyribonuclease activity / dinucleotide insertion or deletion binding / PCNA-p21 complex / mitotic telomere maintenance via semi-conservative replication / Cytosolic iron-sulfur cluster assembly / purine-specific mismatch base pair DNA N-glycosylase activity / positive regulation of DNA-directed DNA polymerase activity / MutLalpha complex binding / nuclear lamina / DNA replication proofreading / Polymerase switching / Telomere C-strand (Lagging Strand) Synthesis / Processive synthesis on the lagging strand / nucleotide-excision repair, DNA gap filling / PCNA complex / 3'-5'-DNA exonuclease activity / Processive synthesis on the C-strand of the telomere / Removal of the Flap Intermediate / 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 / Polymerase switching on the C-strand of the telomere / Removal of the Flap Intermediate from the C-strand / replisome / aggresome / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / DNA strand elongation involved in DNA replication / response to L-glutamate / histone acetyltransferase binding / leading strand elongation / DNA synthesis involved in DNA repair / DNA polymerase processivity factor activity / error-free translesion synthesis / replication fork processing / G1/S-Specific Transcription / DNA biosynthetic process / response to dexamethasone / nuclear replication fork / SUMOylation of DNA replication proteins / estrous cycle / PCNA-Dependent Long Patch Base Excision Repair / mismatch repair / cyclin-dependent protein kinase holoenzyme complex / translesion synthesis / fatty acid homeostasis / positive regulation of DNA replication / error-prone translesion synthesis / response to cadmium ion / DNA polymerase binding / response to UV / base-excision repair, gap-filling / positive regulation of DNA repair / epithelial cell differentiation / positive regulation of endothelial cell proliferation / Translesion synthesis by REV1 / Translesion synthesis by POLK / Translesion synthesis by POLI / Gap-filling DNA repair synthesis and ligation in GG-NER / TP53 Regulates Transcription of Genes Involved in G2 Cell Cycle Arrest / replication fork / male germ cell nucleus / liver regeneration / Recognition of DNA damage by PCNA-containing replication complex / nuclear estrogen receptor binding / Termination of translesion DNA synthesis / Translesion Synthesis by POLH / HDR through Homologous Recombination (HRR) / Dual Incision in GG-NER / DNA-templated DNA replication / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / receptor tyrosine kinase binding / cellular response to hydrogen peroxide / cellular response to UV / protein-macromolecule adaptor activity / cellular response to xenobiotic stimulus / response to estradiol / E3 ubiquitin ligases ubiquitinate target proteins / 4 iron, 4 sulfur cluster binding / heart development / DNA replication / chromosome, telomeric region / damaged DNA binding / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nuclear body / nucleotide binding / DNA repair / centrosome / chromatin binding / chromatin / protein-containing complex binding / enzyme binding / negative regulation of transcription by RNA polymerase II / DNA binding / extracellular exosome
Similarity search - Function
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 / C4-type zinc-finger of DNA polymerase delta / C4-type zinc-finger of DNA polymerase delta ...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 / C4-type zinc-finger of DNA polymerase delta / C4-type zinc-finger of DNA polymerase delta / DNA polymerase delta/II small subunit family / 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 / DNA polymerase family B, thumb domain / DNA polymerase family B signature. / 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-directed DNA polymerase, family B, multifunctional domain / DNA polymerase, palm domain superfamily / DNA polymerase type-B family / DNA-directed DNA polymerase, family B / Ribonuclease H superfamily / Ribonuclease H-like superfamily / DNA/RNA polymerase superfamily
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: 4.86 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
DepositionJun 19, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 25, 2019Provider: repository / Type: Initial release
Revision 1.1Mar 11, 2020Group: Data collection / Database references / Category: citation / citation_author / em_imaging_optics
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 / _em_imaging_optics.phase_plate

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Structure visualization

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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
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area24700 Å2
ΔGint-127 kcal/mol
Surface area115220 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 / Primer (molecular biology)


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 / Iron–sulfur cluster


Mass: 351.640 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe4S4
#10: Chemical ChemComp-TTP / THYMIDINE-5'-TRIPHOSPHATE / Thymidine 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
1PolD holoenzymeCOMPLEX#1-#5, #70MULTIPLE SOURCES
2Human polymerase deltaCOMPLEX#1-#41RECOMBINANT
3Proliferating cell nuclear antigenCOMPLEX#51RECOMBINANT
4DNACOMPLEX#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
SpecimenConc.: 0.355 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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 FIELDBright-field microscopy / Nominal magnification: 75000 X / Calibrated magnification: 130000 X / Nominal defocus max: 600 nm / Nominal defocus min: 400 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: 60 sec. / Electron dose: 35 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 3575
EM imaging opticsPhase plate: VOLTA PHASE PLATE
Image scansSampling size: 14 µm / Width: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
2EPU1.11image acquisition
4Gctf1.18CTF correction
10RELION3initial Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.86 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 32282 / Symmetry type: POINT

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