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- PDB-8ouw: Cryo-EM structure of CMG helicase bound to TIM-1/TIPN-1 and homod... -

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Entry
Database: PDB / ID: 8ouw
TitleCryo-EM structure of CMG helicase bound to TIM-1/TIPN-1 and homodimeric DNSN-1 on fork DNA (Caenorhabditis elegans)
Components
  • (DNA replication complex GINS protein ...) x 2
  • (DNA replication licensing factor ...) x 6
  • (Probable DNA replication complex GINS protein ...) x 2
  • Cell division control protein 45 homolog
  • DNA Lagging Strand Template
  • DNA Leading Strand Template
  • Downstream Neighbor of SoN homolog
  • Protein TIPIN homolog
  • Protein timeless homolog
KeywordsREPLICATION / DNA Replication / DNA Replication Initiation / Replisome / DONSON
Function / homology
Function and homology information


MCM complex assembly / Unwinding of DNA / Assembly of the pre-replicative complex / Orc1 removal from chromatin / Activation of the pre-replicative complex / Switching of origins to a post-replicative state / regulation of sister chromatid cohesion / : / replication fork arrest / gonad development ...MCM complex assembly / Unwinding of DNA / Assembly of the pre-replicative complex / Orc1 removal from chromatin / Activation of the pre-replicative complex / Switching of origins to a post-replicative state / regulation of sister chromatid cohesion / : / replication fork arrest / gonad development / cell cycle phase transition / DNA strand elongation involved in mitotic DNA replication / nuclear DNA replication / negative regulation of DNA helicase activity / GINS complex / mitotic DNA replication preinitiation complex assembly / meiotic sister chromatid cohesion / mitotic DNA replication / DNA replication checkpoint signaling / MCM complex / DNA replication preinitiation complex / double-strand break repair via break-induced replication / locomotion / replication fork protection complex / pronucleus / mitotic DNA replication initiation / mitotic intra-S DNA damage checkpoint signaling / embryo development ending in birth or egg hatching / regulation of DNA-templated DNA replication initiation / DNA strand elongation involved in DNA replication / mitotic sister chromatid cohesion / DNA duplex unwinding / DNA unwinding involved in DNA replication / replication fork processing / DNA replication origin binding / mitotic sister chromatid segregation / DNA replication initiation / condensed chromosome / DNA helicase activity / meiotic cell cycle / helicase activity / DNA-templated DNA replication / single-stranded DNA binding / chromosome / nervous system development / DNA helicase / DNA replication / hydrolase activity / cell cycle / cell division / DNA repair / chromatin binding / positive regulation of cell population proliferation / chromatin / ATP hydrolysis activity / DNA binding / ATP binding / metal ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Donson / Chromosome segregation in meiosis protein 3 / TIPIN/Csm3/Swi3 / Replication Fork Protection Component Swi3 / Timeless / Timeless, N-terminal / Timeless protein / CDC45 family / CDC45-like protein / DNA replication complex GINS protein Psf2 ...Donson / Chromosome segregation in meiosis protein 3 / TIPIN/Csm3/Swi3 / Replication Fork Protection Component Swi3 / Timeless / Timeless, N-terminal / Timeless protein / CDC45 family / CDC45-like protein / DNA replication complex GINS protein Psf2 / 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 / DNA replication licensing factor Mcm5 / 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 / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Nucleic acid-binding, OB-fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / DNA / DNA (> 10) / Protein timeless homolog / Downstream Neighbor of SoN homolog / DNA replication licensing factor MCM7 / Probable DNA replication complex GINS protein PSF2 / DNA replication licensing factor mcm-6 / DNA replication licensing factor mcm-5 / Probable DNA replication complex GINS protein PSF1 ...PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / DNA / DNA (> 10) / Protein timeless homolog / Downstream Neighbor of SoN homolog / DNA replication licensing factor MCM7 / Probable DNA replication complex GINS protein PSF2 / DNA replication licensing factor mcm-6 / DNA replication licensing factor mcm-5 / Probable DNA replication complex GINS protein PSF1 / Cell division control protein 45 homolog / DNA replication licensing factor mcm-4 / DNA replication complex GINS protein PSF3 / Protein TIPIN homolog / DNA replication complex GINS protein SLD5 / DNA replication licensing factor MCM3 / DNA replication licensing factor MCM2
Similarity search - Component
Biological speciesCaenorhabditis elegans (invertebrata)
Saccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.75 Å
AuthorsJenkyn-Bedford, M. / Yeeles, J.T.P. / Labib, K.P.M.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom) United Kingdom
Cancer Research UK United Kingdom
CitationJournal: Science / Year: 2023
Title: DNSN-1 recruits GINS for CMG helicase assembly during DNA replication initiation in .
Authors: Yisui Xia / Remi Sonneville / Michael Jenkyn-Bedford / Liqin Ji / Constance Alabert / Ye Hong / Joseph T P Yeeles / Karim P M Labib /
Abstract: Assembly of the CMG (CDC-45-MCM-2-7-GINS) helicase is the key regulated step during eukaryotic DNA replication initiation. Until now, it was unclear whether metazoa require additional factors that ...Assembly of the CMG (CDC-45-MCM-2-7-GINS) helicase is the key regulated step during eukaryotic DNA replication initiation. Until now, it was unclear whether metazoa require additional factors that are not present in yeast. In this work, we show that DNSN-1, the ortholog of human DONSON, functions during helicase assembly in a complex with MUS-101/TOPBP1. DNSN-1 is required to recruit the GINS complex to chromatin, and a cryo-electron microscopy structure indicates that DNSN-1 positions GINS on the MCM-2-7 helicase motor (comprising the six MCM-2 to MCM-7 proteins), by direct binding of DNSN-1 to GINS and MCM-3, using interfaces that we show are important for initiation and essential for viability. These findings identify DNSN-1 as a missing link in our understanding of DNA replication initiation, suggesting that initiation defects underlie the human disease syndrome that results from DONSON mutations.
History
DepositionApr 25, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 16, 2023Provider: repository / Type: Initial release
Revision 1.1Aug 23, 2023Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Aug 30, 2023Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.3Oct 4, 2023Group: Database references / Category: citation / Item: _citation.journal_volume

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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 mcm-4
5: DNA replication licensing factor mcm-5
6: DNA replication licensing factor mcm-6
7: DNA replication licensing factor MCM7
A: Probable DNA replication complex GINS protein PSF1
B: Probable 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 homolog
F: Downstream Neighbor of SoN homolog
G: Downstream Neighbor of SoN homolog
H: Downstream Neighbor of SoN homolog
I: DNA Leading Strand Template
J: DNA Leading Strand Template
K: Protein timeless homolog
L: Protein TIPIN homolog
M: DNA Lagging Strand Template
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,153,73432
Polymers1,151,28519
Non-polymers2,44913
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA replication licensing factor ... , 6 types, 6 molecules 234567

#1: Protein DNA replication licensing factor MCM2


Mass: 99448.586 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Caenorhabditis elegans (invertebrata) / Gene: mcm-2, CELE_Y17G7B.5, Y17G7B.5 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q9XXI9, DNA helicase
#2: Protein DNA replication licensing factor MCM3


Mass: 90810.664 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Caenorhabditis elegans (invertebrata) / Gene: mcm-3, C25D7.6, CELE_C25D7.6 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q9XVR7, DNA helicase
#3: Protein DNA replication licensing factor mcm-4


Mass: 91687.555 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Caenorhabditis elegans (invertebrata) / Gene: mcm-4, let-358, lin-6, Y39G10AR.14 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q95XQ8, DNA helicase
#4: Protein DNA replication licensing factor mcm-5


Mass: 85057.734 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Caenorhabditis elegans (invertebrata) / Gene: mcm-5, R10E4.4 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q21902, DNA helicase
#5: Protein DNA replication licensing factor mcm-6


Mass: 91248.320 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Caenorhabditis elegans (invertebrata) / Gene: mcm-6, ZK632.1 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P34647, DNA helicase
#6: Protein DNA replication licensing factor MCM7


Mass: 81721.352 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Caenorhabditis elegans (invertebrata) / Gene: mcm-7, MCM7, CELE_F32D1.10, F32D1.10 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: O16297, DNA helicase

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Probable DNA replication complex GINS protein ... , 2 types, 2 molecules AB

#7: Protein Probable DNA replication complex GINS protein PSF1 / GINS complex subunit 1


Mass: 23064.361 Da / Num. of mol.: 1
Mutation: Protease-cleaved N-terminal expression tag (Gly-Pro-Gly-Ser)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Caenorhabditis elegans (invertebrata) / Gene: psf-1, R53.6 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q22019
#8: Protein Probable DNA replication complex GINS protein PSF2 / GINS complex subunit 2


Mass: 20349.330 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Caenorhabditis elegans (invertebrata) / Gene: psf-2, F31C3.5 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: O62193

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DNA replication complex GINS protein ... , 2 types, 2 molecules CD

#9: Protein DNA replication complex GINS protein PSF3


Mass: 21717.668 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Caenorhabditis elegans (invertebrata) / Gene: psf-3, CELE_Y65B4BR.8, Y65B4BR.8 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q9BL54
#10: Protein DNA replication complex GINS protein SLD5


Mass: 25705.123 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Caenorhabditis elegans (invertebrata) / Gene: sld-5, CELE_Y113G7B.24, Y113G7B.24 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q9U2W9

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Protein , 4 types, 6 molecules EFGHKL

#11: Protein Cell division control protein 45 homolog


Mass: 66245.758 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Caenorhabditis elegans (invertebrata) / Gene: evl-18, CELE_F34D10.2, F34D10.2 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q7JMR0
#12: Protein Downstream Neighbor of SoN homolog


Mass: 66079.383 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Caenorhabditis elegans (invertebrata) / Gene: dnsn-1, C24H12.5, CELE_C24H12.5 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: H1ZUV7
#14: Protein Protein timeless homolog


Mass: 157256.656 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Caenorhabditis elegans (invertebrata) / Gene: tim-1, csg-5, Y75B8A.22 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: G5EDN3
#15: Protein Protein TIPIN homolog / CSM3 homolog


Mass: 27415.215 Da / Num. of mol.: 1
Mutation: Protease-cleaved N-terminal expression tag (Gly-Pro-Gly-Ser)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Caenorhabditis elegans (invertebrata) / Gene: tipn-1, F23C8.9 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q9TXI0

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DNA chain , 2 types, 3 molecules IJM

#13: DNA chain DNA Leading Strand Template


Mass: 26396.836 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae (brewer's yeast)
#16: DNA chain DNA Lagging Strand Template


Mass: 18524.887 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae (brewer's yeast)

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Non-polymers , 3 types, 13 molecules

#17: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Zn
#18: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg
#19: Chemical
ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Comment: AMP-PNP, energy-carrying molecule analogue*YM

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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

ComponentName: Complex of CMG helicase bound to TIM-1/TIPN-1 and homodimeric DNSN-1 on fork DNA (Caenorhabditis elegans)
Type: COMPLEX / Entity ID: #1-#16 / Source: RECOMBINANT
Source (natural)Organism: Caenorhabditis elegans (invertebrata)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.5
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

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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 defocus max: 2500 nm / Nominal defocus min: 1000 nm
Image recordingAverage exposure time: 1.7 sec. / Electron dose: 40.1 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
2EPU2image acquisition
13PHENIX1.19model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.75 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 33900 / Symmetry type: POINT

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