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- EMDB-47925: Consensus refinement of yeast CMG G4 stall state 1 -

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Basic information

Entry
Database: EMDB / ID: EMD-47925
TitleConsensus refinement of yeast CMG G4 stall state 1
Map dataConsensus refinement of yeast CMG stall state 1
Sample
  • Complex: Yeast CMG-CTM G4 stall state 1
Keywordshelicase / DNA replication / cell division / fork stalling / translocation / REPLICATION
Biological speciesSaccharomyces cerevisiae W303 (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsAllwein B / Batra S / Remus D / Hite R
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM152094 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM126907 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)P30 CA008748 United States
CitationJournal: Science / Year: 2025
Title: G-quadruplex-stalled eukaryotic replisome structure reveals helical inchworm DNA translocation.
Authors: Sahil Batra / Benjamin Allwein / Charanya Kumar / Sujan Devbhandari / Jan-Gert Brüning / Soon Bahng / Chong M Lee / Kenneth J Marians / Richard K Hite / Dirk Remus /
Abstract: DNA G-quadruplexes (G4s) are non-B-form DNA secondary structures that threaten genome stability by impeding DNA replication. To elucidate how G4s induce replication fork arrest, we characterized fork ...DNA G-quadruplexes (G4s) are non-B-form DNA secondary structures that threaten genome stability by impeding DNA replication. To elucidate how G4s induce replication fork arrest, we characterized fork collisions with preformed G4s in the parental DNA using reconstituted yeast and human replisomes. We demonstrate that a single G4 in the leading strand template is sufficient to stall replisomes by arresting the CMG helicase. Cryo-electron microscopy structures of stalled yeast and human CMG complexes reveal that the folded G4 is lodged inside the central CMG channel, arresting translocation. The G4 stabilizes the CMG at distinct translocation intermediates, suggesting an unprecedented helical inchworm mechanism for DNA translocation. These findings illuminate the eukaryotic replication fork mechanism under normal and perturbed conditions.
History
DepositionNov 14, 2024-
Header (metadata) releaseMar 26, 2025-
Map releaseMar 26, 2025-
UpdateMar 26, 2025-
Current statusMar 26, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_47925.png

Downloads & links

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Map

FileDownload / File: emd_47925.map.gz / Format: CCP4 / Size: 343 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationConsensus refinement of yeast CMG stall state 1
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 448 pix.
= 370.048 Å		0.83 Å/pix.
x 448 pix.
= 370.048 Å		0.83 Å/pix.
x 448 pix.
= 370.048 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.826 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.225
Minimum - Maximum-0.54844624 - 1.1402695
Average (Standard dev.)0.00061609864 (±0.036529325)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions448448448
Spacing448448448
CellA=B=C: 370.04797 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_47925_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Density-modified consensus refinement of yeast CMG stall state 1

Fileemd_47925_additional_1.map
AnnotationDensity-modified consensus refinement of yeast CMG stall state 1
Projections & Slices
AxesZYX

Projections

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Density Histograms

Histogram

Histogram (log scale)

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Half map: Consensus refinement half-map A of yeast CMG stall state 1

Fileemd_47925_half_map_1.map
AnnotationConsensus refinement half-map A of yeast CMG stall state 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Consensus refinement half-map B of yeast CMG stall state 1

Fileemd_47925_half_map_2.map
AnnotationConsensus refinement half-map B of yeast CMG stall state 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Yeast CMG-CTM G4 stall state 1

EntireName: Yeast CMG-CTM G4 stall state 1
Components
  • Complex: Yeast CMG-CTM G4 stall state 1

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Supramolecule #1: Yeast CMG-CTM G4 stall state 1

SupramoleculeName: Yeast CMG-CTM G4 stall state 1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#15
Source (natural)Organism: Saccharomyces cerevisiae W303 (yeast)
Molecular weightTheoretical: 186.08 KDa

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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

Concentration.01 mg/mL
BufferpH: 7.6
Component:
ConcentrationFormulaName
20.0 mMC8H17KN2O4SHEPES-KOH
100.0 mMCH3CO2KPotassium acetate
10.0 mMMg(CH3COO)2Magnesium acetate
2.5 mMC4H10O2S2DTT
0.02 %H(C2H4O)nO(C6H4)C9H19NP-40S
100.0 uMC10H17N6O12P3AMP-PNP
5.0 mMC10H16N5O13P3ATP
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Support film - Film thickness: 0.5
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: Wait time 30 seconds after sample application; blot time 30 seconds with blot force 0.

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 3975 / Average exposure time: 3.0 sec. / Average electron dose: 66.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 29000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1895342
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.0.0) / Number images used: 66410
Initial angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 4.0.0)
Details: Stochastic gradient descent (cryoSPARC ab initio model generation)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.0.0)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 4.0.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: Other / Chain - Initial model type: in silico model / Details: ModelAngelo
DetailsInitial fitting was performed de novo by ModelAngelo, then iteratively improved with ChimeraX/ISOLDE, Coot, and Phenix real-space refinement algorithms.
RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 79.47

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