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- EMDB-16916: Bipartite interaction of TOPBP1 with the GINS complex -

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Basic information

Entry
Database: EMDB / ID: EMD-16916
TitleBipartite interaction of TOPBP1 with the GINS complex
Map data
Sample
  • Complex: GINS complex bound to TOPBP1
    • Protein or peptide: DNA replication complex GINS protein PSF1
    • Protein or peptide: DNA replication complex GINS protein PSF2
    • Protein or peptide: DNA replication complex GINS protein PSF3
    • Protein or peptide: DNA replication complex GINS protein SLD5
    • Protein or peptide: Topoisomerase (DNA) II binding protein 1
KeywordsScaffold / Replisome / Recruitment / REPLICATION
Function / homology
Function and homology information


Unwinding of DNA / DNA strand elongation involved in mitotic DNA replication / GINS complex / CMG complex / double-strand break repair via break-induced replication / mitotic DNA replication initiation / inner cell mass cell proliferation / DNA unwinding involved in DNA replication / isomerase activity / nucleoplasm ...Unwinding of DNA / DNA strand elongation involved in mitotic DNA replication / GINS complex / CMG complex / double-strand break repair via break-induced replication / mitotic DNA replication initiation / inner cell mass cell proliferation / DNA unwinding involved in DNA replication / isomerase activity / nucleoplasm / nucleus / cytoplasm
Similarity search - Function
TopBP1, first BRCT domain / Secretoglobin superfamily / 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 ...TopBP1, first BRCT domain / Secretoglobin superfamily / 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 / BRCA1 C Terminus (BRCT) domain / breast cancer carboxy-terminal domain / BRCT domain profile. / BRCT domain / BRCT domain superfamily
Similarity search - Domain/homology
Topoisomerase (DNA) II binding protein 1 / DNA replication complex GINS protein PSF1 / DNA replication complex GINS protein SLD5 / DNA replication complex GINS protein PSF3 / DNA replication complex GINS protein PSF2
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsDay M / Oliver AW / Pearl LH
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Cancer Research UK United Kingdom
CitationJournal: Nat Commun / Year: 2024
Title: TopBP1 utilises a bipartite GINS binding mode to support genome replication.
Authors: Matthew Day / Bilal Tetik / Milena Parlak / Yasser Almeida-Hernández / Markus Räschle / Farnusch Kaschani / Heike Siegert / Anika Marko / Elsa Sanchez-Garcia / Markus Kaiser / Isabel A ...Authors: Matthew Day / Bilal Tetik / Milena Parlak / Yasser Almeida-Hernández / Markus Räschle / Farnusch Kaschani / Heike Siegert / Anika Marko / Elsa Sanchez-Garcia / Markus Kaiser / Isabel A Barker / Laurence H Pearl / Antony W Oliver / Dominik Boos /
Abstract: Activation of the replicative Mcm2-7 helicase by loading GINS and Cdc45 is crucial for replication origin firing, and as such for faithful genetic inheritance. Our biochemical and structural studies ...Activation of the replicative Mcm2-7 helicase by loading GINS and Cdc45 is crucial for replication origin firing, and as such for faithful genetic inheritance. Our biochemical and structural studies demonstrate that the helicase activator GINS interacts with TopBP1 through two separate binding surfaces, the first involving a stretch of highly conserved amino acids in the TopBP1-GINI region, the second a surface on TopBP1-BRCT4. The two surfaces bind to opposite ends of the A domain of the GINS subunit Psf1. Mutation analysis reveals that either surface is individually able to support TopBP1-GINS interaction, albeit with reduced affinity. Consistently, either surface is sufficient for replication origin firing in Xenopus egg extracts and becomes essential in the absence of the other. The TopBP1-GINS interaction appears sterically incompatible with simultaneous binding of DNA polymerase epsilon (Polε) to GINS when bound to Mcm2-7-Cdc45, although TopBP1-BRCT4 and the Polε subunit PolE2 show only partial competitivity in binding to Psf1. Our TopBP1-GINS model improves the understanding of the recently characterised metazoan pre-loading complex. It further predicts the coordination of three molecular origin firing processes, DNA polymerase epsilon arrival, TopBP1 ejection and GINS integration into Mcm2-7-Cdc45.
History
DepositionMar 26, 2023-
Header (metadata) releaseMar 13, 2024-
Map releaseMar 13, 2024-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16916.png

Downloads & links

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Map

FileDownload / File: emd_16916.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.74 Å/pix.
x 280 pix.
= 207.2 Å		0.74 Å/pix.
x 280 pix.
= 207.2 Å		0.74 Å/pix.
x 280 pix.
= 207.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.74 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.003
Minimum - Maximum-0.021244345 - 0.03759489
Average (Standard dev.)0.00003447198 (±0.0012448505)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 207.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #2

Fileemd_16916_additional_1.map
Projections & Slices
AxesZYX

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Additional map: #1

Fileemd_16916_additional_2.map
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Half map: #2

Fileemd_16916_half_map_1.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_16916_half_map_2.map
Projections & Slices
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Sample components

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Entire : GINS complex bound to TOPBP1

EntireName: GINS complex bound to TOPBP1
Components
  • Complex: GINS complex bound to TOPBP1
    • Protein or peptide: DNA replication complex GINS protein PSF1
    • Protein or peptide: DNA replication complex GINS protein PSF2
    • Protein or peptide: DNA replication complex GINS protein PSF3
    • Protein or peptide: DNA replication complex GINS protein SLD5
    • Protein or peptide: Topoisomerase (DNA) II binding protein 1

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Supramolecule #1: GINS complex bound to TOPBP1

SupramoleculeName: GINS complex bound to TOPBP1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: DNA replication complex GINS protein PSF1

MacromoleculeName: DNA replication complex GINS protein PSF1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 17.772438 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
MFCEKAMELI RELHRAPEGQ LPAFNEDGLR QVLEEMKALY EQNQSDVNEA KSGGRSDLIP TIKFRHCSLL RNRRCTVAYL YDRLLRIRA LRWEYGSILP NALRFHMAAE EMEWFNNYKR SLATYMRSLG GDEGLDITQD MKPPKSLYIE VR

UniProtKB: DNA replication complex GINS protein PSF1

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Macromolecule #2: DNA replication complex GINS protein PSF2

MacromoleculeName: DNA replication complex GINS protein PSF2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 21.453713 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
MDAAEVEFLA EKELVTIIPN FSLDKIYLIG GDLGPFNPGL PVEVPLWLAI NLKQRQKCRL LPPEWMDVEK LEKMRDHERK EETFTPMPS PYYMELTKLL LNHASDNIPK ADEIRTLVKD MWDTRIAKLR VSADSFVRQQ EAHAKLDNLT LMEINTSGTF L TQALNHMY KLRTNLQPLE STQSQDF

UniProtKB: DNA replication complex GINS protein PSF2

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Macromolecule #3: DNA replication complex GINS protein PSF3

MacromoleculeName: DNA replication complex GINS protein PSF3 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 24.562611 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MSEAYFRVES GALGPEENFL SLDDILMSHE KLPVRTETAM PRLGAFFLER SAGAETDNAV PQGSKLELPL WLAKGLFDNK RRILSVELP KIYQEGWRTV FSADPNVVDL HKMGPHFYGF GSQLLHFDSP ENADISQSLL QTFIGRFRRI MDSSQNAYNE D TSALVARL ...String:
MSEAYFRVES GALGPEENFL SLDDILMSHE KLPVRTETAM PRLGAFFLER SAGAETDNAV PQGSKLELPL WLAKGLFDNK RRILSVELP KIYQEGWRTV FSADPNVVDL HKMGPHFYGF GSQLLHFDSP ENADISQSLL QTFIGRFRRI MDSSQNAYNE D TSALVARL DEMERGLFQT GQKGLNDFQC WEKGQASQIT ASNLVQNYKK RKFTDMED

UniProtKB: DNA replication complex GINS protein PSF3

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Macromolecule #4: DNA replication complex GINS protein SLD5

MacromoleculeName: DNA replication complex GINS protein SLD5 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 30.831814 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MHHHHHHGRM DYKDDDDKAD YKDDDDKADY KDDDDKGRPM TEEVDFLGQD SDGGSEEVVL TPAELIERLE QAWMNEKFAP ELLESKPEI VECVMEQLEH MEENLRRAKR EDLKVSIHQM EMERIRYVLS SYLRCRLMKI EKFFPHVLEK EKTRPEGEPS S LSPEELAF ...String:
MHHHHHHGRM DYKDDDDKAD YKDDDDKADY KDDDDKGRPM TEEVDFLGQD SDGGSEEVVL TPAELIERLE QAWMNEKFAP ELLESKPEI VECVMEQLEH MEENLRRAKR EDLKVSIHQM EMERIRYVLS SYLRCRLMKI EKFFPHVLEK EKTRPEGEPS S LSPEELAF AREFMANTES YLKNVALKHM PPNLQKVDLF RAVPKPDLDS YVFLRVRERQ ENILVEPDTD EQRDYVIDLE KG SQHLIRY KTIAPLVASG AVQLI

UniProtKB: DNA replication complex GINS protein SLD5

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Macromolecule #5: Topoisomerase (DNA) II binding protein 1

MacromoleculeName: Topoisomerase (DNA) II binding protein 1 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 52.397535 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MGSHHHHHHG SLEVLFQGPH MASNSLNSKL EPTLENLENL DVSAFQAPED LLDGCRIYLC GFSGRKLDKL RRLINSGGGV RFNQLNEDV THVIVGDYDD ELKQFWNKSA HRPHVVGAKW LLECFSKGYM LSEEPYIHAN YQPVEIPVSH QPESKAALLK K KNSSFSKK ...String:
MGSHHHHHHG SLEVLFQGPH MASNSLNSKL EPTLENLENL DVSAFQAPED LLDGCRIYLC GFSGRKLDKL RRLINSGGGV RFNQLNEDV THVIVGDYDD ELKQFWNKSA HRPHVVGAKW LLECFSKGYM LSEEPYIHAN YQPVEIPVSH QPESKAALLK K KNSSFSKK DFAPSEKHEQ ADEDLLSQYE NGSSTVVEAK TSEARPFNDS THAEPLNDST HISLQEENQS SVSHCVPDVS TI TEEGLFS QKSFLVLGFS NENESNIANI IKENAGKIMS LLSRTVADYA VVPLLGCEVE ATVGEVVTNT WLVTCIDYQT LFD PKSNPL FTPVPVMTGM TPLEDCVISF SQCAGAEKES LTFLANLLGA SVQEYFVRKS NAKKGMFAST HLILKERGGS KYEA AKKWN LPAVTIAWLL ETARTGKRAD ESHFLIENST KEERSLETEI TNGINLNSDT AEHPRRAWSH PQFEK

UniProtKB: Topoisomerase (DNA) II binding protein 1

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.2 µm
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 39.69290657 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 154278

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