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- EMDB-50392: Structure of a eukaryotic replisome stalled at a lagging-strand T... -

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Basic information

Entry
Database: EMDB / ID: EMD-50392
TitleStructure of a eukaryotic replisome stalled at a lagging-strand Topoisomerase 1 cleavage complex with Tof1-Csm3
Map data
Sample
  • Complex: Budding yeast replisome stalled at a leading-strand Topoisomerase 1 cleavage complex.
KeywordsDNA / polymerase / epsilon / PCNA / leading strand / human / replication / replisome / proofrerading
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.2 Å
AuthorsWesthorpe R / Roske JJ / Yeeles JTP
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
UK Research and Innovation (UKRI) United Kingdom
CitationJournal: Mol Cell / Year: 2024
Title: Mechanisms controlling replication fork stalling and collapse at topoisomerase 1 cleavage complexes.
Authors: Rose Westhorpe / Johann J Roske / Joseph T P Yeeles /
Abstract: Topoisomerase 1 cleavage complexes (Top1-ccs) comprise a DNA-protein crosslink and a single-stranded DNA break that can significantly impact the DNA replication machinery (replisome). Consequently, ...Topoisomerase 1 cleavage complexes (Top1-ccs) comprise a DNA-protein crosslink and a single-stranded DNA break that can significantly impact the DNA replication machinery (replisome). Consequently, inhibitors that trap Top1-ccs are used extensively in research and clinical settings to generate DNA replication stress, yet how the replisome responds upon collision with a Top1-cc remains obscure. By reconstituting collisions between budding yeast replisomes, assembled from purified proteins, and site-specific Top1-ccs, we have uncovered mechanisms underlying replication fork stalling and collapse. We find that stalled replication forks are surprisingly stable and that their stability is influenced by the template strand that Top1 is crosslinked to, the fork protection complex proteins Tof1-Csm3 (human TIMELESS-TIPIN), and the convergence of replication forks. Moreover, nascent-strand mapping and cryoelectron microscopy (cryo-EM) of stalled forks establishes replisome remodeling as a key factor in the initial response to Top1-ccs. These findings have important implications for the use of Top1 inhibitors in research and in the clinic.
History
DepositionMay 23, 2024-
Header (metadata) releaseSep 11, 2024-
Map releaseSep 11, 2024-
UpdateOct 2, 2024-
Current statusOct 2, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50392.png

Downloads & links

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Map

FileDownload / File: emd_50392.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.86 Å/pix.
x 288 pix.
= 535.68 Å		1.86 Å/pix.
x 288 pix.
= 535.68 Å		1.86 Å/pix.
x 288 pix.
= 535.68 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.86 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.14
Minimum - Maximum-0.67954046 - 1.2593898
Average (Standard dev.)0.003650344 (±0.033479374)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 535.68 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Refined consensus, globally sharpened

Fileemd_50392_additional_1.map
AnnotationRefined consensus, globally sharpened
Projections & Slices
AxesZYX

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Half map: #1

Fileemd_50392_half_map_1.map
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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_50392_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

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Histogram (log scale)

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Sample components

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Entire : Budding yeast replisome stalled at a leading-strand Topoisomerase...

EntireName: Budding yeast replisome stalled at a leading-strand Topoisomerase 1 cleavage complex.
Components
  • Complex: Budding yeast replisome stalled at a leading-strand Topoisomerase 1 cleavage complex.

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Supramolecule #1: Budding yeast replisome stalled at a leading-strand Topoisomerase...

SupramoleculeName: Budding yeast replisome stalled at a leading-strand Topoisomerase 1 cleavage complex.
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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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

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 39.9 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 5886
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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