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- EMDB-52107: S. cerevisiae CMG, Ctf4, T-C, DNA. Local refinement following sig... -

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Basic information

Entry
Database: EMDB / ID: EMD-52107
TitleS. cerevisiae CMG, Ctf4, T-C, DNA. Local refinement following signal subtraction
Map dataConsensus local Refinement. CMG, Ctf4, Tof1-Csm3, DNA.
Sample
  • Complex: Complex of S. cerevisiae CMG, Tof1-Csm3, fork DNA, Ctf4, Pol epsilon, Ctf18-RFC
KeywordsDNA / Replication / CMG / Ctf18-RFC / replisome
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.34 Å
AuthorsYeeles JTP / Jones ML / Fletcher EE
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
UK Research and Innovation (UKRI) United Kingdom
CitationJournal: EMBO J / Year: 2025
Title: Competition for the nascent leading strand shapes the requirements for PCNA loading in the replisome.
Authors: Emma E Fletcher / Morgan L Jones / Joseph T P Yeeles /
Abstract: During DNA replication, the DNA polymerases Pol δ and Pol ε utilise the ring-shaped sliding clamp PCNA to enhance their processivity. PCNA loading onto DNA is accomplished by the clamp loaders RFC ...During DNA replication, the DNA polymerases Pol δ and Pol ε utilise the ring-shaped sliding clamp PCNA to enhance their processivity. PCNA loading onto DNA is accomplished by the clamp loaders RFC and Ctf18-RFC, which function primarily on the lagging and the leading strand, respectively. RFC activity is essential for lagging-strand replication by Pol δ, but it is unclear why Ctf18-RFC is required for leading-strand PCNA loading and why RFC cannot fulfil this function. Here, we show that RFC cannot load PCNA once Pol ε has been incorporated into the budding yeast replisome and commenced leading-strand synthesis, and this state is maintained during replisome progression. By contrast, we find that Ctf18-RFC is uniquely equipped to load PCNA onto the leading strand and show that this activity requires a direct interaction between Ctf18 and the CMG (Cdc45-MCM-GINS) helicase. Our work uncovers a mechanistic basis for why replisomes require a dedicated leading-strand clamp loader.
History
DepositionNov 15, 2024-
Header (metadata) releaseFeb 26, 2025-
Map releaseFeb 26, 2025-
UpdateOct 8, 2025-
Current statusOct 8, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_52107.png

Downloads & links

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Map

FileDownload / File: emd_52107.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationConsensus local Refinement. CMG, Ctf4, Tof1-Csm3, DNA.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.37 Å/pix.
x 440 pix.
= 604.516 Å		1.37 Å/pix.
x 440 pix.
= 604.516 Å		1.37 Å/pix.
x 440 pix.
= 604.516 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.3739 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.846
Minimum - Maximum-1.4900016 - 4.069584
Average (Standard dev.)0.004446123 (±0.09014351)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 604.51605 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_52107_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Locally filtered map of local refinement. CMG, Ctf4, Tof1-Csm3, DNA.

Fileemd_52107_additional_1.map
AnnotationLocally filtered map of local refinement. CMG, Ctf4, Tof1-Csm3, DNA.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Sharpened, local refinement. CMG, Ctf4, Tof1-Csm3, DNA.

Fileemd_52107_additional_2.map
AnnotationSharpened, local refinement. CMG, Ctf4, Tof1-Csm3, DNA.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A, local refinement. CMG, Ctf4, Tof1-Csm3, DNA.

Fileemd_52107_half_map_1.map
AnnotationHalf map A, local refinement. CMG, Ctf4, Tof1-Csm3, DNA.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B, local refinement. CMG, Ctf4, Tof1-Csm3, DNA.

Fileemd_52107_half_map_2.map
AnnotationHalf map B, local refinement. CMG, Ctf4, Tof1-Csm3, DNA.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of S. cerevisiae CMG, Tof1-Csm3, fork DNA, Ctf4, Pol epsi...

EntireName: Complex of S. cerevisiae CMG, Tof1-Csm3, fork DNA, Ctf4, Pol epsilon, Ctf18-RFC
Components
  • Complex: Complex of S. cerevisiae CMG, Tof1-Csm3, fork DNA, Ctf4, Pol epsilon, Ctf18-RFC

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Supramolecule #1: Complex of S. cerevisiae CMG, Tof1-Csm3, fork DNA, Ctf4, Pol epsi...

SupramoleculeName: Complex of S. cerevisiae CMG, Tof1-Csm3, fork DNA, Ctf4, Pol epsilon, Ctf18-RFC
type: complex / ID: 1 / Parent: 0 / Details: Sample prepared using GraFix
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: 7.6
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 36.8901 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: 1.2 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER / Details: Ab-initio reconstruction
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.34 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 47823
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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