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- PDB-7pmk: S. cerevisiae replisome-SCF(Dia2) complex bound to double-strande... -

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Basic information

Entry
Database: PDB / ID: 7pmk
TitleS. cerevisiae replisome-SCF(Dia2) complex bound to double-stranded DNA (conformation I)
Components
  • (DNA polymerase epsilon ...) x 2
  • (DNA replication complex GINS protein ...) x 4
  • (DNA replication licensing factor ...) x 5
  • Cell division control protein 45,Cell division control protein 45
  • Chromosome segregation in meiosis protein 3
  • DNA helicase
  • DNA polymerase alpha-binding protein
  • E3 ubiquitin ligase complex SCF subunit
  • Lagging strand template DNA
  • Leading strand template DNA
  • Protein DIA2
  • Topoisomerase 1-associated factor 1
KeywordsREPLICATION / Genome stability / DNA replication / Ubiquitination / termination / replisome / cryo-EM / CMG / SCF(Dia2)
Function / homology
Function and homology information


maintenance of DNA repeat elements / DNA-templated DNA replication maintenance of fidelity / gene conversion / Unwinding of DNA / invasive growth in response to glucose limitation / replication fork arrest / DNA replication initiation / protein-containing complex disassembly / meiotic chromosome segregation / epsilon DNA polymerase complex ...maintenance of DNA repeat elements / DNA-templated DNA replication maintenance of fidelity / gene conversion / Unwinding of DNA / invasive growth in response to glucose limitation / replication fork arrest / DNA replication initiation / protein-containing complex disassembly / meiotic chromosome segregation / epsilon DNA polymerase complex / MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / MCM complex binding / GINS complex / DNA strand elongation involved in mitotic DNA replication / nuclear DNA replication / mitotic DNA replication preinitiation complex assembly / premeiotic DNA replication / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / mitotic DNA replication / SUMO binding / Activation of the pre-replicative complex / CMG complex / establishment of mitotic sister chromatid cohesion / DNA replication checkpoint signaling / single-stranded 3'-5' DNA helicase activity / entrainment of circadian clock / single-stranded DNA 3'-5' DNA exonuclease activity / nuclear pre-replicative complex / MCM complex / Activation of ATR in response to replication stress / DNA replication preinitiation complex / Termination of translesion DNA synthesis / replication fork protection complex / mitotic DNA replication checkpoint signaling / mitotic DNA replication initiation / double-strand break repair via break-induced replication / mitotic intra-S DNA damage checkpoint signaling / silent mating-type cassette heterochromatin formation / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / single-stranded DNA helicase activity / SCF ubiquitin ligase complex / nucleotide-excision repair, DNA gap filling / DNA replication proofreading / mitotic sister chromatid cohesion / DNA strand elongation involved in DNA replication / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / leading strand elongation / replication fork processing / regulation of DNA replication / DNA unwinding involved in DNA replication / nuclear replication fork / 3'-5' DNA helicase activity / DNA replication origin binding / cullin family protein binding / Dual incision in TC-NER / subtelomeric heterochromatin formation / DNA replication initiation / error-prone translesion synthesis / DNA helicase activity / helicase activity / base-excision repair, gap-filling / meiotic cell cycle / replication fork / base-excision repair / DNA-templated DNA replication / double-strand break repair via nonhomologous end joining / double-strand break repair / mitotic cell cycle / single-stranded DNA binding / 4 iron, 4 sulfur cluster binding / ubiquitin-dependent protein catabolic process / double-stranded DNA binding / DNA replication / DNA helicase / chromosome, telomeric region / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / hydrolase activity / protein ubiquitination / cell cycle / DNA repair / nucleotide binding / mRNA binding / chromatin binding / ATP hydrolysis activity / DNA binding / zinc ion binding / nucleoplasm / ATP binding / nucleus / metal ion binding / cytoplasm / cytosol
Similarity search - Function
F-box domain / Chromosome segregation in meiosis protein 3 / TIPIN/Csm3/Swi3 / Replication Fork Protection Component Swi3 / Timeless / Timeless, N-terminal / Timeless protein / DNA polymerase epsilon, subunit B / Minichromosome loss protein Mcl1, middle region / Minichromosome loss protein, Mcl1, middle region ...F-box domain / Chromosome segregation in meiosis protein 3 / TIPIN/Csm3/Swi3 / Replication Fork Protection Component Swi3 / Timeless / Timeless, N-terminal / Timeless protein / DNA polymerase epsilon, subunit B / Minichromosome loss protein Mcl1, middle region / Minichromosome loss protein, Mcl1, middle region / DNA polymerase epsilon, catalytic subunit A, C-terminal / DNA polymerase epsilon catalytic subunit / Domain of unknown function (DUF1744) / DUF1744 / Leucine-rich repeat, cysteine-containing subtype / Leucine-rich repeat - CC (cysteine-containing) subfamily / A Receptor for Ubiquitination Targets / CDC45 family / DNA replication complex GINS protein Psf2 / CDC45-like protein / GINS complex, subunit Psf1 / GINS complex, subunit Psf3 / GINS complex, subunit Psf3 superfamily / F-box domain profile. / DNA replication complex GINS protein SLD5, C-terminal / GINS, helical bundle-like domain superfamily / GINS complex protein Sld5, alpha-helical domain / DNA replication complex GINS protein SLD5 C-terminus / GINS complex subunit Sld5 / GINS subunit, domain A / GINS complex protein helical bundle domain / F-box-like domain superfamily / SKP1 component, dimerisation / S-phase kinase-associated protein 1 / SKP1-like, dimerisation domain superfamily / Skp1 family, dimerisation domain / MCM4, winged helix domain / DNA polymerase alpha/delta/epsilon, subunit B / DNA replication licensing factor Mcm5 / DNA polymerase alpha/epsilon subunit B / F-box domain / DNA replication licensing factor Mcm3 / Mini-chromosome maintenance complex protein 4 / DNA replication licensing factor Mcm6 / DNA replication licensing factor Mcm7 / Mcm6, C-terminal winged-helix domain / MCM6 C-terminal winged-helix domain / DNA replication licensing factor Mcm2 / Mini-chromosome maintenance protein 2 / Mini-chromosome maintenance, conserved site / MCM family signature. / MCM N-terminal domain / MCM N-terminal domain / MCM OB domain / MCM OB domain / Mini-chromosome maintenance protein / MCM, AAA-lid domain / MCM P-loop domain / MCM AAA-lid domain / MCM family domain profile. / minichromosome maintenance proteins / MCM domain / Leucine Rich Repeat / DNA polymerase family B, thumb domain / S-phase kinase-associated protein 1-like / SKP1 component, POZ domain / Skp1 family, tetramerisation domain / Found in Skp1 protein family / 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 / TPR repeat region circular profile. / Leucine-rich repeat profile. / SKP1/BTB/POZ domain superfamily / Leucine-rich repeat / Leucine-rich repeat domain superfamily / Tetratricopeptide-like helical domain superfamily / Ribonuclease H superfamily / Ribonuclease H-like superfamily / WD40-repeat-containing domain superfamily / Winged helix-like DNA-binding domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Nucleic acid-binding, OB-fold / DNA/RNA polymerase superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / DNA / DNA (> 10) / DNA (> 100) / DNA replication licensing factor MCM5 / DNA replication complex GINS protein PSF2 / DNA replication licensing factor MCM2 / DNA replication complex GINS protein PSF1 / E3 ubiquitin ligase complex SCF subunit / DNA replication licensing factor MCM7 ...PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / DNA / DNA (> 10) / DNA (> 100) / DNA replication licensing factor MCM5 / DNA replication complex GINS protein PSF2 / DNA replication licensing factor MCM2 / DNA replication complex GINS protein PSF1 / E3 ubiquitin ligase complex SCF subunit / DNA replication licensing factor MCM7 / DNA polymerase alpha-binding protein / DNA replication complex GINS protein PSF3 / DNA polymerase epsilon catalytic subunit A / DNA replication licensing factor MCM3 / DNA polymerase epsilon subunit B / DNA replication licensing factor MCM4 / DNA replication licensing factor MCM6 / Topoisomerase 1-associated factor 1 / DNA replication complex GINS protein SLD5 / Chromosome segregation in meiosis protein 3 / Cell division control protein 45 / Protein DIA2
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsJenkyn-Bedford, M. / Yeeles, J.T.P. / Deegan, T.D.
Funding support United Kingdom, 3items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MC_UP_1201/12 United Kingdom
Medical Research Council (MRC, United Kingdom)MC_UU_12016/13 United Kingdom
Wellcome Trust204678/Z/16/Z United Kingdom
CitationJournal: Nature / Year: 2021
Title: A conserved mechanism for regulating replisome disassembly in eukaryotes.
Authors: Michael Jenkyn-Bedford / Morgan L Jones / Yasemin Baris / Karim P M Labib / Giuseppe Cannone / Joseph T P Yeeles / Tom D Deegan /
Abstract: Replisome disassembly is the final step of eukaryotic DNA replication and is triggered by ubiquitylation of the CDC45-MCM-GINS (CMG) replicative helicase. Despite being driven by evolutionarily ...Replisome disassembly is the final step of eukaryotic DNA replication and is triggered by ubiquitylation of the CDC45-MCM-GINS (CMG) replicative helicase. Despite being driven by evolutionarily diverse E3 ubiquitin ligases in different eukaryotes (SCF in budding yeast, CUL2 in metazoa), replisome disassembly is governed by a common regulatory principle, in which ubiquitylation of CMG is suppressed before replication termination, to prevent replication fork collapse. Recent evidence suggests that this suppression is mediated by replication fork DNA. However, it is unknown how SCF and CUL2 discriminate terminated from elongating replisomes, to selectively ubiquitylate CMG only after termination. Here we used cryo-electron microscopy to solve high-resolution structures of budding yeast and human replisome-E3 ligase assemblies. Our structures show that the leucine-rich repeat domains of Dia2 and LRR1 are structurally distinct, but bind to a common site on CMG, including the MCM3 and MCM5 zinc-finger domains. The LRR-MCM interaction is essential for replisome disassembly and, crucially, is occluded by the excluded DNA strand at replication forks, establishing the structural basis for the suppression of CMG ubiquitylation before termination. Our results elucidate a conserved mechanism for the regulation of replisome disassembly in eukaryotes, and reveal a previously unanticipated role for DNA in preserving replisome integrity.
History
DepositionSep 2, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 10, 2021Provider: repository / Type: Initial release
Revision 2.0Nov 17, 2021Group: Atomic model / Data collection ...Atomic model / Data collection / Derived calculations / Other / Source and taxonomy
Category: atom_site / database_PDB_rev ...atom_site / database_PDB_rev / database_PDB_rev_record / em_admin / ndb_struct_na_base_pair / ndb_struct_na_base_pair_step / pdbx_data_processing_status / pdbx_database_proc / pdbx_database_status / pdbx_entity_src_syn / pdbx_nonpoly_scheme / pdbx_seq_map_depositor_info / pdbx_struct_sheet_hbond / struct_conf / struct_conf_type / struct_conn / struct_conn_type / struct_sheet / struct_sheet_order / struct_sheet_range
Item: _atom_site.auth_seq_id / _em_admin.last_update ..._atom_site.auth_seq_id / _em_admin.last_update / _pdbx_database_status.pdb_format_compatible / _pdbx_entity_src_syn.ncbi_taxonomy_id / _pdbx_entity_src_syn.organism_scientific / _pdbx_nonpoly_scheme.pdb_seq_num / _pdbx_seq_map_depositor_info.one_letter_code_mod
Revision 2.1Jan 5, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title / _citation_author.identifier_ORCID
Revision 2.2Jan 26, 2022Group: Source and taxonomy / Category: entity_src_gen
Item: _entity_src_gen.gene_src_strain / _entity_src_gen.pdbx_gene_src_ncbi_taxonomy_id / _entity_src_gen.pdbx_gene_src_scientific_name
Revision 2.3Feb 2, 2022Group: Source and taxonomy / Category: pdbx_entity_src_syn / Item: _pdbx_entity_src_syn.ncbi_taxonomy_id
Revision 2.4Jul 17, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

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Assembly

Deposited unit
2: DNA replication licensing factor MCM2
3: DNA replication licensing factor MCM3
4: DNA replication licensing factor MCM4
5: DNA helicase
6: DNA replication licensing factor MCM6
7: DNA replication licensing factor MCM7
A: DNA replication complex GINS protein PSF1
B: DNA replication complex GINS protein PSF2
C: DNA replication complex GINS protein PSF3
D: DNA replication complex GINS protein SLD5
E: Cell division control protein 45,Cell division control protein 45
F: DNA polymerase alpha-binding protein
G: DNA polymerase alpha-binding protein
H: DNA polymerase alpha-binding protein
I: Leading strand template DNA
J: Lagging strand template DNA
K: E3 ubiquitin ligase complex SCF subunit
L: Protein DIA2
Q: DNA polymerase epsilon catalytic subunit A
R: DNA polymerase epsilon subunit B
X: Topoisomerase 1-associated factor 1
Y: Chromosome segregation in meiosis protein 3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,811,92935
Polymers1,809,88022
Non-polymers2,04913
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: equilibrium centrifugation
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA replication licensing factor ... , 5 types, 5 molecules 23467

#1: Protein DNA replication licensing factor MCM2 / Minichromosome maintenance protein 2


Mass: 98911.539 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PACBIOSEQ_LOCUS187, PACBIOSEQ_LOCUS193, PACBIOSEQ_LOCUS195, PACBIOSEQ_LOCUS196, SCNYR20_0007007400, SCP684_0007007100
Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A6A5Q1S9, DNA helicase
#2: Protein DNA replication licensing factor MCM3 / Minichromosome maintenance protein 3


Mass: 111987.562 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: MCM3, YEL032W, SYGP-ORF23 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P24279, DNA helicase
#3: Protein DNA replication licensing factor MCM4 / Cell division control protein 54


Mass: 105138.375 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: MCM4, CDC54, HCD21, YPR019W, YP9531.13 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P30665, DNA helicase
#5: Protein DNA replication licensing factor MCM6 / Minichromosome maintenance protein 6


Mass: 113110.211 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: MCM6, YGL201C / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P53091, DNA helicase
#6: Protein DNA replication licensing factor MCM7


Mass: 95049.875 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: MCM7, PACBIOSEQ_LOCUS429 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A6A5Q4N0, DNA helicase

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Protein , 7 types, 9 molecules 5EFGHKLXY

#4: Protein DNA helicase


Mass: 86505.734 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PACBIOSEQ_LOCUS4054, PACBIOSEQ_LOCUS4112, PACBIOSEQ_LOCUS4129, PACBIOSEQ_LOCUS4153, PACBIOSEQ_LOCUS4202, SCNYR20_0004029000, SCP684_0004028600
Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A6A5PUY8, DNA helicase
#11: Protein Cell division control protein 45,Cell division control protein 45


Mass: 75154.703 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: CDC45, SLD4, YLR103C, L8004.11 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q08032
#12: Protein DNA polymerase alpha-binding protein


Mass: 108610.148 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: SCNYR20_0005036600 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A6A5Q5Y5
#15: Protein E3 ubiquitin ligase complex SCF subunit


Mass: 22357.270 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PACBIOSEQ_LOCUS1258, PACBIOSEQ_LOCUS1259, PACBIOSEQ_LOCUS1261, PACBIOSEQ_LOCUS1263, PACBIOSEQ_LOCUS1270, PACBIOSEQ_LOCUS1277, PACBIOSEQ_LOCUS1282, SCNYR20_0001050400, SCP684_0001049800
Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A6A5Q435
#16: Protein Protein DIA2 / Digs into agar protein 2


Mass: 85368.844 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: DIA2, YOR080W, YOR29-31 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q08496
#19: Protein Topoisomerase 1-associated factor 1


Mass: 141296.875 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: TOF1, YNL273W, N0636 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P53840
#20: Protein Chromosome segregation in meiosis protein 3


Mass: 36588.758 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: CSM3, YMR048W, YM9796.01 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q04659

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DNA replication complex GINS protein ... , 4 types, 4 molecules ABCD

#7: Protein DNA replication complex GINS protein PSF1


Mass: 24230.576 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PACBIOSEQ_LOCUS944, PACBIOSEQ_LOCUS956, PACBIOSEQ_LOCUS958, SCNYR20_0001022500, SCP684_0001022000
Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A6A5Q203
#8: Protein DNA replication complex GINS protein PSF2


Mass: 25096.807 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PACBIOSEQ_LOCUS3163, PACBIOSEQ_LOCUS3191, PACBIOSEQ_LOCUS3224, PACBIOSEQ_LOCUS3231, PACBIOSEQ_LOCUS3255, SCNYR20_0009012300, SCP684_0009011800
Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A6A5PX40
#9: Protein DNA replication complex GINS protein PSF3


Mass: 22004.160 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PACBIOSEQ_LOCUS5150, PACBIOSEQ_LOCUS5239, PACBIOSEQ_LOCUS5244, PACBIOSEQ_LOCUS5270, PACBIOSEQ_LOCUS5331
Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A6L1BWJ4
#10: Protein DNA replication complex GINS protein SLD5


Mass: 33983.617 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: SLD5, YDR489W / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q03406

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DNA chain , 2 types, 2 molecules IJ

#13: DNA chain Leading strand template DNA


Mass: 37688.645 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Synthetic construct (others)
#14: DNA chain Lagging strand template DNA


Mass: 35259.238 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Synthetic construct (others)

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DNA polymerase epsilon ... , 2 types, 2 molecules QR

#17: Protein DNA polymerase epsilon catalytic subunit A / 3'-5' exodeoxyribonuclease / DNA polymerase II subunit A


Mass: 255890.422 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: POL2, DUN2, YNL262W, N0825 / Production host: Saccharomyces cerevisiae (brewer's yeast)
References: UniProt: P21951, DNA-directed DNA polymerase, Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters
#18: Protein DNA polymerase epsilon subunit B / DNA polymerase II subunit 2


Mass: 78425.852 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: DPB2, YPR175W, P9705.7 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P24482

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Non-polymers , 3 types, 13 molecules

#21: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#22: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#23: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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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
IDNameTypeDetailsEntity IDParent-IDSource
1Budding yeast replisome on double-stranded DNA engaged with SCF(Dia2) (conformation I)COMPLEXComplex reconstituted in vitro from purified proteins and DNA#1-#200RECOMBINANT
2DNACOMPLEX#13-#141RECOMBINANT
3ReplisomeCOMPLEX#1-#12, #17-#201RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
23Saccharomyces cerevisiae (brewer's yeast)4932
32DNA molecule (others)2853804
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
23Saccharomyces cerevisiae (brewer's yeast)4932
32synthetic construct (others)32630
Buffer solutionpH: 7.6
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 15 mA / Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R2/2
VitrificationCryogen name: ETHANE / Details: Manual plunger

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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: 81000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 400 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 4 sec. / Electron dose: 38.8 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 12730
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
2EPU2image acquisition
4RELION3CTF correction
10RELION3initial Euler assignment
11RELION3final Euler assignment
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2160000
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 369254
Details: Composite map produced from individual cryo-EM density maps (resolutions 3.2 - 4.0 A) using Phenix combine_focused_maps. See publication for details.
Symmetry type: POINT

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