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- PDB-8p5e: S. cerevisiae nexus-sCMGE after DNA replication initiation -

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Basic information

Entry
Database: PDB / ID: 8p5e
TitleS. cerevisiae nexus-sCMGE after DNA replication initiation
Components
  • (DNA polymerase epsilon ...) x 2
  • (DNA replication complex GINS protein ...) x 4
  • (DNA replication licensing factor ...) x 5
  • Cell division control protein 45
  • DNA (19-MER)
  • DNA (7-MER)
  • Minichromosome maintenance protein 5
KeywordsREPLICATION / Saccharomyces cerevisiae / helicase / CMGE / initiation of DNA replication / DNA / DNA unwinding
Function / homology
Function and homology information


DNA-templated DNA replication maintenance of fidelity / gene conversion / Unwinding of DNA / DNA replication initiation / 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-templated DNA replication maintenance of fidelity / gene conversion / Unwinding of DNA / DNA replication initiation / 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 / single-stranded 3'-5' DNA helicase activity / 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 / regulation of DNA-templated DNA replication initiation / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / single-stranded DNA helicase activity / nucleotide-excision repair, DNA gap filling / DNA replication proofreading / mitotic sister chromatid cohesion / DNA strand elongation involved in DNA replication / leading strand elongation / DNA unwinding involved in DNA replication / nuclear replication fork / 3'-5' DNA helicase activity / DNA replication origin binding / Dual incision in TC-NER / subtelomeric heterochromatin formation / DNA replication initiation / error-prone translesion synthesis / heterochromatin formation / helicase activity / base-excision repair, gap-filling / 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 / double-stranded DNA binding / DNA helicase / chromosome, telomeric region / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / cell cycle / nucleotide binding / mRNA binding / DNA damage response / chromatin binding / ATP hydrolysis activity / DNA binding / zinc ion binding / nucleoplasm / ATP binding / nucleus / metal ion binding / cytoplasm
Similarity search - Function
DNA polymerase epsilon, subunit B / DNA polymerase epsilon, catalytic subunit A, C-terminal / DNA polymerase epsilon catalytic subunit / Domain of unknown function (DUF1744) / DUF1744 / CDC45 family / DNA replication complex GINS protein Psf2 / CDC45-like protein / GINS complex, subunit Psf1 / GINS complex, subunit Psf3 ...DNA polymerase epsilon, subunit B / DNA polymerase epsilon, catalytic subunit A, C-terminal / DNA polymerase epsilon catalytic subunit / Domain of unknown function (DUF1744) / DUF1744 / CDC45 family / DNA replication complex GINS protein Psf2 / CDC45-like protein / GINS complex, subunit Psf1 / GINS complex, subunit Psf3 / GINS complex, subunit Psf3 superfamily / 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 / MCM4, winged helix domain / DNA polymerase alpha/delta/epsilon, subunit B / DNA replication licensing factor Mcm5 / DNA polymerase alpha/epsilon subunit B / 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 / DNA polymerase family B, thumb domain / 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 / Winged helix-like DNA-binding 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
ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / 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 ...ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / 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 / DNA replication licensing factor MCM6 / DNA replication complex GINS protein SLD5 / Cell division control protein 45 / DNA replication complex GINS protein PSF3 / 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: 3.9 Å
AuthorsHenrikus, S.S. / Willhoft, O.
Funding support United Kingdom, European Union, France, 4items
OrganizationGrant numberCountry
The Francis Crick InstituteFC001065 and FC001066 United Kingdom
European Research Council (ERC)grant agreement no. 820102European Union
Human Frontier Science Program (HFSP)LT000834/2020-L France
European Molecular Biology Organization (EMBO)ALTF 962-2019European Union
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: Unwinding of a eukaryotic origin of replication visualized by cryo-EM.
Authors: Sarah S Henrikus / Marta H Gross / Oliver Willhoft / Thomas Pühringer / Jacob S Lewis / Allison W McClure / Julia F Greiwe / Giacomo Palm / Andrea Nans / John F X Diffley / Alessandro Costa /
Abstract: To prevent detrimental chromosome re-replication, DNA loading of a double hexamer of the minichromosome maintenance (MCM) replicative helicase is temporally separated from DNA unwinding. Upon S-phase ...To prevent detrimental chromosome re-replication, DNA loading of a double hexamer of the minichromosome maintenance (MCM) replicative helicase is temporally separated from DNA unwinding. Upon S-phase transition in yeast, DNA unwinding is achieved in two steps: limited opening of the double helix and topological separation of the two DNA strands. First, Cdc45, GINS and Polε engage MCM to assemble a double CMGE with two partially separated hexamers that nucleate DNA melting. In the second step, triggered by Mcm10, two CMGEs separate completely, eject the lagging-strand template and cross paths. To understand Mcm10 during helicase activation, we used biochemical reconstitution with cryogenic electron microscopy. We found that Mcm10 splits the double CMGE by engaging the N-terminal homo-dimerization face of MCM. To eject the lagging strand, DNA unwinding is started from the N-terminal side of MCM while the hexamer channel becomes too narrow to harbor duplex DNA.
History
DepositionMay 24, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 29, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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 (19-MER)
B: DNA (7-MER)
C: DNA replication complex GINS protein PSF3
D: DNA replication complex GINS protein SLD5
E: Cell division control protein 45
F: DNA polymerase epsilon subunit B
G: DNA polymerase epsilon catalytic subunit A
H: DNA replication complex GINS protein PSF1
I: DNA replication complex GINS protein PSF2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,140,44231
Polymers1,137,02815
Non-polymers3,41416
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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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: 111720.242 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 , 2 types, 2 molecules 5E

#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


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

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DNA chain , 2 types, 2 molecules AB

#7: DNA chain DNA (19-MER)


Mass: 5905.973 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#8: DNA chain DNA (7-MER)


Mass: 2084.392 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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

#9: Protein DNA replication complex GINS protein PSF3 / Partner of Sld five 3


Mass: 25718.070 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PSF3, YOL146W / Production host: Escherichia coli (E. coli) / References: UniProt: Q12146
#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: Escherichia coli (E. coli) / References: UniProt: Q03406
#14: Protein DNA replication complex GINS protein PSF1 / Partner of Sld five 1


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: PSF1, YDR013W, PZA208, YD8119.18 / Production host: Escherichia coli (E. coli) / References: UniProt: Q12488
#15: Protein DNA replication complex GINS protein PSF2 / Partner of Sld five 2


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: PSF2, YJL072C, HRF213, J1086 / Production host: Escherichia coli (E. coli) / References: UniProt: P40359

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

#12: 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
#13: Protein DNA polymerase epsilon catalytic subunit A / 3'-5' exodeoxyribonuclease / DNA polymerase II subunit A


Mass: 255992.484 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

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Non-polymers , 4 types, 16 molecules

#16: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#17: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#18: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#19: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM

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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
IDNameTypeEntity IDParent-IDSource
1Complex of Mcm2-7, Cdc45, GINS (Psf1-3, Sld5) DNA polymerase epsilon (subunit A and B), 19-mer DNA molecule with complementary 7-mer DNA moleculeCOMPLEX#1-#150MULTIPLE SOURCES
2DNA replication complex GINS containing Psf1, Psf2, Psf3 and Sld5COMPLEX#9-#10, #14-#151RECOMBINANT
3DNA replication licensing factors MCM2-7COMPLEX#1-#61RECOMBINANT
4DNA polymerase epsilon contains catalytic subunit A and non-catalytic subunit BCOMPLEX#12-#131RECOMBINANT
5DNACOMPLEX#7-#81RECOMBINANT
6Cdc45COMPLEX#111RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Saccharomyces cerevisiae (brewer's yeast)4932
32Saccharomyces cerevisiae (brewer's yeast)4932
43Saccharomyces cerevisiae (brewer's yeast)4932
54Saccharomyces cerevisiae (brewer's yeast)4932
65synthetic construct (others)32630
76Saccharomyces cerevisiae (brewer's yeast)4932
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Saccharomyces cerevisiae (brewer's yeast)4932
32Escherichia coli (E. coli)562
43Saccharomyces cerevisiae (brewer's yeast)4932
54Saccharomyces cerevisiae (brewer's yeast)4932
65synthetic construct (others)32630
76Saccharomyces cerevisiae (brewer's yeast)4932
Buffer solutionpH: 7.6
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3300 nm / Nominal defocus min: 1800 nm
Image recordingElectron dose: 1.67 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.21_5207 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 85107 / Symmetry type: POINT

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