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- PDB-6tnz: Human polymerase delta-FEN1-PCNA toolbelt -

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Basic information

Entry
Database: PDB / ID: 6tnz
TitleHuman polymerase delta-FEN1-PCNA toolbelt
Components
  • (DNA polymerase delta subunit ...) x 3
  • DNA polymerase delta catalytic subunit
  • DNA primer
  • DNA template
  • Flap endonuclease 1
  • Proliferating cell nuclear antigen
KeywordsREPLICATION / Protein
Function / homology
Function and homology information


delta DNA polymerase complex / positive regulation of sister chromatid cohesion / DNA synthesis involved in UV-damage excision repair / flap endonuclease activity / nucleotide-excision repair complex / zeta DNA polymerase complex / telomere maintenance via semi-conservative replication / double-stranded DNA exodeoxyribonuclease activity / positive regulation of deoxyribonuclease activity / dinucleotide insertion or deletion binding ...delta DNA polymerase complex / positive regulation of sister chromatid cohesion / DNA synthesis involved in UV-damage excision repair / flap endonuclease activity / nucleotide-excision repair complex / zeta DNA polymerase complex / telomere maintenance via semi-conservative replication / double-stranded DNA exodeoxyribonuclease activity / positive regulation of deoxyribonuclease activity / dinucleotide insertion or deletion binding / PCNA-p21 complex / Cytosolic iron-sulfur cluster assembly / mitotic telomere maintenance via semi-conservative replication / nucleic acid metabolic process / purine-specific mismatch base pair DNA N-glycosylase activity / 5'-flap endonuclease activity / nuclear lamina / MutLalpha complex binding / DNA replication, removal of RNA primer / positive regulation of DNA-directed DNA polymerase activity / Polymerase switching / Telomere C-strand (Lagging Strand) Synthesis / 5'-3' exonuclease activity / Processive synthesis on the lagging strand / UV protection / PCNA complex / Removal of the Flap Intermediate / Processive synthesis on the C-strand of the telomere / Mismatch repair (MMR) directed by MSH2:MSH3 (MutSbeta) / Polymerase switching on the C-strand of the telomere / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / Transcription of E2F targets under negative control by DREAM complex / Removal of the Flap Intermediate from the C-strand / HDR through MMEJ (alt-NHEJ) / replisome / aggresome / exonuclease activity / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / nucleotide-excision repair, DNA gap filling / response to L-glutamate / DNA replication proofreading / 3'-5'-DNA exonuclease activity / DNA strand elongation involved in DNA replication / histone acetyltransferase binding / DNA synthesis involved in DNA repair / error-free translesion synthesis / DNA polymerase processivity factor activity / DNA biosynthetic process / G1/S-Specific Transcription / leading strand elongation / response to dexamethasone / replication fork processing / Early Phase of HIV Life Cycle / nuclear replication fork / SUMOylation of DNA replication proteins / POLB-Dependent Long Patch Base Excision Repair / estrous cycle / PCNA-Dependent Long Patch Base Excision Repair / cyclin-dependent protein kinase holoenzyme complex / fatty acid homeostasis / error-prone translesion synthesis / mismatch repair / translesion synthesis / response to cadmium ion / DNA polymerase binding / response to UV / positive regulation of endothelial cell proliferation / epithelial cell differentiation / positive regulation of DNA repair / Translesion synthesis by REV1 / Translesion synthesis by POLK / Translesion synthesis by POLI / Gap-filling DNA repair synthesis and ligation in GG-NER / base-excision repair, gap-filling / TP53 Regulates Transcription of Genes Involved in G2 Cell Cycle Arrest / replication fork / positive regulation of DNA replication / male germ cell nucleus / liver regeneration / nuclear estrogen receptor binding / Recognition of DNA damage by PCNA-containing replication complex / Termination of translesion DNA synthesis / Translesion Synthesis by POLH / double-strand break repair via homologous recombination / HDR through Homologous Recombination (HRR) / Dual Incision in GG-NER / DNA-templated DNA replication / receptor tyrosine kinase binding / memory / cellular response to hydrogen peroxide / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / cellular response to UV / double-strand break repair / RNA-DNA hybrid ribonuclease activity / cellular response to xenobiotic stimulus / E3 ubiquitin ligases ubiquitinate target proteins / response to estradiol / protein-macromolecule adaptor activity / heart development
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 / Flap endonuclease 1 / DNA polymerase delta/II small subunit family ...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 / Flap endonuclease 1 / DNA polymerase delta/II small subunit family / C4-type zinc-finger of DNA polymerase delta / C4-type zinc-finger of DNA polymerase delta / XPG protein signature 2. / XPG conserved site / XPG protein signature 1. / XPG/Rad2 endonuclease / XPG, N-terminal / XPG-I domain / XPG N-terminal domain / XPG I-region / Xeroderma pigmentosum G I-region / Xeroderma pigmentosum G N-region / 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 / Helix-hairpin-helix motif, class 2 / Helix-hairpin-helix class 2 (Pol1 family) motifs / 5'-3' exonuclease, C-terminal domain superfamily / DNA polymerase family B, thumb domain / DNA polymerase family B signature. / DNA-directed DNA polymerase, family B, conserved site / PIN-like domain superfamily / : / 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 / Flap endonuclease 1 / 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: 4.05 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: 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
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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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-10540
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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
H: Flap endonuclease 1
P: DNA primer
T: DNA template
hetero molecules


Theoretical massNumber of molelcules
Total (without water)394,05113
Polymers393,15210
Non-polymers8993
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area27570 Å2
ΔGint-150 kcal/mol
Surface area128400 Å2
MethodPISA

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Components

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Protein , 3 types, 5 molecules AEFGH

#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
#6: Protein Flap endonuclease 1 / FEN-1 / DNase IV / Flap structure-specific endonuclease 1 / Maturation factor 1 / hFEN-1


Mass: 42617.039 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: FEN1, RAD2 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P39748, Hydrolases; Acting on ester bonds

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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

#7: DNA chain DNA primer


Mass: 7665.987 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#8: 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

#9: Chemical ChemComp-ZN / ZINC ION


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


Mass: 351.640 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe4S4
#11: 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-#6, #80MULTIPLE SOURCES
2DNA polymeraseCOMPLEX#1-#41RECOMBINANT
3Proliferating cell nuclear antigen, flap endonucleaseCOMPLEX#5-#61RECOMBINANT
4DNA primer, templateCOMPLEX#7-#81RECOMBINANT
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
EM imaging opticsPhase plate: OTHER
Image scansWidth: 11520 / Height: 8184

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
4CTFFIND4.1CTF correction
11RELION3.1final Euler assignment
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.05 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 47291 / Symmetry type: POINT

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