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

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Basic information

Entry
Database: PDB / ID: 7pmn
TitleS. cerevisiae replisome-SCF(Dia2) complex bound to double-stranded DNA (conformation II)
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 polymerase alpha-binding protein
  • Lagging strand template DNA
  • Leading strand template DNA
  • Minichromosome maintenance protein 5
  • Protein DIA2
  • Suppressor of kinetochore protein 1
  • Topoisomerase 1-associated factor 1
KeywordsREPLICATION / Genome stability / DNA replication / Ubiquitination / termination / replisome / cryo-EM / CMG / SCF(Dia2)
Function / homology
Function and homology information


RAVE complex / Iron uptake and transport / CBF3 complex / regulation of transcription by galactose / : / cellular response to methylmercury / establishment of sister chromatid cohesion / vacuolar proton-transporting V-type ATPase complex assembly / mitotic DNA replication termination / gene conversion ...RAVE complex / Iron uptake and transport / CBF3 complex / regulation of transcription by galactose / : / cellular response to methylmercury / establishment of sister chromatid cohesion / vacuolar proton-transporting V-type ATPase complex assembly / mitotic DNA replication termination / gene conversion / septin ring assembly / DNA-templated DNA replication maintenance of fidelity / maintenance of DNA repeat elements / Unwinding of DNA / invasive growth in response to glucose limitation / replication fork protection complex / replication fork arrest / DNA replication initiation / Cul8-RING ubiquitin ligase complex / meiotic chromosome segregation / epsilon DNA polymerase complex / DNA strand elongation involved in mitotic DNA replication / MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / GINS complex / MCM complex binding / mitotic DNA replication preinitiation complex assembly / protein-containing complex disassembly / nuclear DNA replication / premeiotic DNA replication / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / nucleotide-excision repair, DNA gap filling / regulation of exit from mitosis / SUMO binding / Activation of the pre-replicative complex / kinetochore assembly / mitotic DNA replication / DNA replication checkpoint signaling / establishment of mitotic sister chromatid cohesion / nuclear pre-replicative complex / CMG complex / DNA replication proofreading / : / DNA replication preinitiation complex / single-stranded DNA 3'-5' DNA exonuclease activity / Activation of ATR in response to replication stress / positive regulation of D-glucose transmembrane transport / mitotic DNA replication checkpoint signaling / vacuolar acidification / double-strand break repair via break-induced replication / MCM complex / exit from mitosis / mitotic DNA replication initiation / protein neddylation / mitochondrial fusion / mitotic intra-S DNA damage checkpoint signaling / silent mating-type cassette heterochromatin formation / single-stranded DNA helicase activity / regulation of DNA-templated DNA replication initiation / mitotic sister chromatid cohesion / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis / SCF ubiquitin ligase complex / DNA strand elongation involved in DNA replication / Ubiquitin-Mediated Degradation of Phosphorylated Cdc25A / Orc1 removal from chromatin / leading strand elongation / nuclear chromosome / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / nuclear replication fork / regulation of DNA replication / Antigen processing: Ubiquitination & Proteasome degradation / Dual incision in TC-NER / replication fork processing / DNA replication origin binding / error-prone translesion synthesis / cullin family protein binding / regulation of protein-containing complex assembly / DNA replication initiation / subtelomeric heterochromatin formation / base-excision repair, gap-filling / negative regulation of cytoplasmic translation / endomembrane system / regulation of mitotic cell cycle / replication fork / DNA helicase activity / helicase activity / meiotic cell cycle / transcription elongation by RNA polymerase II / G1/S transition of mitotic cell cycle / base-excision repair / kinetochore / DNA-templated DNA replication / double-strand break repair via nonhomologous end joining / G2/M transition of mitotic cell cycle / peroxisome / double-strand break repair / mitotic cell cycle / heterochromatin formation
Similarity search - Function
F-box domain / Chromosome segregation in meiosis protein 3 / TIPIN/Csm3/Swi3 / Replication Fork Protection Component Swi3 / Timeless, N-terminal / Timeless / Timeless protein / DNA polymerase epsilon, subunit B / : / DNA polymerase alpha-binding protein Ctf4, C-terminal domain ...F-box domain / Chromosome segregation in meiosis protein 3 / TIPIN/Csm3/Swi3 / Replication Fork Protection Component Swi3 / Timeless, N-terminal / Timeless / Timeless protein / DNA polymerase epsilon, subunit B / : / DNA polymerase alpha-binding protein Ctf4, C-terminal domain / Minichromosome loss protein Mcl1, middle region / Minichromosome loss protein, Mcl1, middle region / : / : / DNA polymerase epsilon catalytic subunit A, thumb domain / Zinc finger domain of DNA polymerase-epsilon / Zinc finger domain of DNA polymerase-epsilon / DNA polymerase epsilon, catalytic subunit A, C-terminal / DNA polymerase epsilon catalytic subunit / Domain of unknown function (DUF1744) / DUF1744 / : / PSF2 N-terminal domain / : / PSF1 C-terminal domain / DNA replication complex GINS protein Psf2 / CDC45 family / CDC45 / GINS complex, subunit Psf3 / DNA replication complex GINS protein SLD5, C-terminal / GINS complex, subunit Psf3 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 / GINS complex, subunit Psf1 / : / MCM3 winged helix domain / GINS, helical bundle-like domain superfamily / Leucine-rich repeat, cysteine-containing subtype / Leucine-rich repeat - CC (cysteine-containing) subfamily / A Receptor for Ubiquitination Targets / F-box domain profile. / DNA polymerase alpha/delta/epsilon, subunit B / DNA polymerase alpha/epsilon subunit B / F-box-like domain superfamily / SKP1 component, dimerisation / S-phase kinase-associated protein 1 / SKP1-like, dimerisation domain superfamily / Skp1 family, dimerisation domain / : / MCM5, C-terminal domain / DNA replication licensing factor MCM7, winged helix / DNA replication licensing factor Mcm5 / MCM4, winged helix domain / DNA replication licensing factor Mcm3 / Mini-chromosome maintenance complex protein 4 / : / MCM3-like, winged helix domain / DNA replication licensing factor Mcm6 / DNA replication licensing factor Mcm7 / Mcm6, C-terminal winged-helix domain / MCM6 C-terminal winged-helix domain / F-box 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 C-terminal AAA(+) ATPase domain (MCM-CTD) profile. / minichromosome maintenance proteins / MCM domain / 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, 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 / 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 / Winged helix-like DNA-binding domain superfamily / WD40-repeat-containing domain superfamily
Similarity search - Domain/homology
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / DNA / DNA (> 10) / DNA (> 100) / 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 MCM2 / Minichromosome maintenance protein 5 ...PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / DNA / DNA (> 10) / DNA (> 100) / 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 MCM2 / Minichromosome maintenance protein 5 / DNA replication licensing factor MCM4 / DNA replication licensing factor MCM7 / DNA replication complex GINS protein PSF2 / Suppressor of kinetochore protein 1 / DNA replication licensing factor MCM6 / Topoisomerase 1-associated factor 1 / DNA polymerase alpha-binding protein / DNA replication complex GINS protein SLD5 / Chromosome segregation in meiosis protein 3 / Cell division control protein 45 / Protein DIA2 / DNA replication complex GINS protein PSF1
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 1.1Nov 17, 2021Group: Data collection / Source and taxonomy
Category: em_admin / pdbx_database_proc ...em_admin / pdbx_database_proc / pdbx_entity_src_syn / pdbx_seq_map_depositor_info
Item: _em_admin.last_update / _pdbx_entity_src_syn.ncbi_taxonomy_id ..._em_admin.last_update / _pdbx_entity_src_syn.ncbi_taxonomy_id / _pdbx_entity_src_syn.organism_scientific / _pdbx_seq_map_depositor_info.one_letter_code_mod
Revision 1.2Jan 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 1.3Jan 26, 2022Group: Source and taxonomy / Category: em_entity_assembly_recombinant / entity_src_gen
Item: _em_entity_assembly_recombinant.ncbi_tax_id / _em_entity_assembly_recombinant.organism ..._em_entity_assembly_recombinant.ncbi_tax_id / _em_entity_assembly_recombinant.organism / _entity_src_gen.gene_src_strain / _entity_src_gen.pdbx_gene_src_ncbi_taxonomy_id / _entity_src_gen.pdbx_gene_src_scientific_name
Revision 1.4Jul 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: Minichromosome maintenance protein 5
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: Suppressor of kinetochore protein 1
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)1,811,44833
Polymers1,809,93022
Non-polymers1,51911
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: MCM2, YBL023C, YBL0438 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P29469, 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 / Cell division control protein 47 / Minichromosome maintenance protein 7


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, CDC47, YBR202W, YBR1441 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P38132, DNA helicase

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Protein , 7 types, 9 molecules 5EFGHKLXY

#4: Protein Minichromosome maintenance protein 5 / Cell division control protein 46


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: MCM5, CDC46, YLR274W, L9328.1 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P29496, 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 / Chromosome replication protein CHL15 / Chromosome transmission fidelity protein 4 / Protein POB1


Mass: 108610.148 Da / Num. of mol.: 3 / Fragment: Mcm6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: CTF4, CHL15, POB1, YPR135W, P9659.7 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q01454
#15: Protein Suppressor of kinetochore protein 1 / Centromere DNA-binding protein complex CBF3 subunit D / E3 ubiquitin ligase complex SCF subunit SKP1


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: SKP1, CBF3D, YDR328C, D9798.14 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P52286
#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 / Partner of Sld five 1


Mass: 24230.576 Da / Num. of mol.: 1 / Fragment: Tof1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PSF1, YDR013W, PZA208, YD8119.18 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q12488
#8: Protein DNA replication complex GINS protein PSF2 / Partner of Sld five 2


Mass: 25096.807 Da / Num. of mol.: 1 / Fragment: Csm3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PSF2, YJL072C, HRF213, J1086 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P40359
#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: 37738.676 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: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Production host: Saccharomyces cerevisiae (brewer's yeast)

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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, 11 molecules

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


Mass: 506.196 Da / Num. of mol.: 2 / 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.: 2 / 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
IDNameTypeDetails (eV)Entity IDParent-IDSource
1Budding yeast replisome on double-stranded DNA engaged with SCF(Dia2) (conformation II)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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