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- EMDB-52505: Polymerase epsilon local refinement in the absence of Ctf18-RFC -

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Basic information

Entry
Database: EMDB / ID: EMD-52505
TitlePolymerase epsilon local refinement in the absence of Ctf18-RFC
Map dataConsensus
Sample
  • Complex: Pol epsilon focussed refinement.
KeywordsReplication / DNA / polymerase / helicase / MCM / CMG
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.25 Å
AuthorsJones ML / Yeeles JTP / Fletcher EE
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom) 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
DepositionJan 9, 2025-
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_52505.png

Downloads & links

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Map

FileDownload / File: emd_52505.map.gz / Format: CCP4 / Size: 1 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationConsensus
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.93 Å/pix.
x 650 pix.
= 604.5 Å		0.93 Å/pix.
x 650 pix.
= 604.5 Å		0.93 Å/pix.
x 650 pix.
= 604.5 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.93 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.257
Minimum - Maximum-0.49752876 - 1.3176755
Average (Standard dev.)-0.0026551129 (±0.02414665)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions650650650
Spacing650650650
CellA=B=C: 604.5 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_52505_msk_1.map
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AxesZYX

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Additional map: Locally filtered

Fileemd_52505_additional_1.map
AnnotationLocally filtered
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Additional map: Sharpened

Fileemd_52505_additional_2.map
AnnotationSharpened
Projections & Slices
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Half map: Half A

Fileemd_52505_half_map_1.map
AnnotationHalf A
Projections & Slices
AxesZYX

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Histogram

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Half map: Half B

Fileemd_52505_half_map_2.map
AnnotationHalf B
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Sample components

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Entire : Pol epsilon focussed refinement.

EntireName: Pol epsilon focussed refinement.
Components
  • Complex: Pol epsilon focussed refinement.

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Supramolecule #1: Pol epsilon focussed refinement.

SupramoleculeName: Pol epsilon focussed refinement. / type: complex / ID: 1 / Parent: 0
Details: Replisome complex comprised of CMG, Csm3-Tof, forked DNA, Ctf4, Ctf18-RFC, Pol epsilon and Mrc1.
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: 7.8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (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: 3.5 µm / Nominal defocus min: 1.5 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
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.25 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 20462
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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