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- EMDB-13988: S. cerevisiae CMGE dimer nucleating origin DNA melting -

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Basic information

Entry
Database: EMDB / ID: EMD-13988
TitleS. cerevisiae CMGE dimer nucleating origin DNA melting
Map datacryoSPARC trans CMGE dimer
Sample
  • Complex: Ternary complex of the CMG helicase melting origin DNA
KeywordsDNA replication / helicase / initiation / DNA origin / REPLICATION
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.2 Å
AuthorsLewis JS / Sousa JS / Costa A
Funding supportEuropean Union, 10 items
OrganizationGrant numberCountry
European Molecular Biology Organization (EMBO)211-2020European Union
H2020 Marie Curie Actions of the European CommissionEuropean Union
European Molecular Biology Organization (EMBO)1177-2020European Union
Human Frontier Science Program (HFSP)LT000834/2020-LEuropean Union
European Molecular Biology Organization (EMBO)962-2019European Union
The Francis Crick InstituteFC001065European Union
The Francis Crick InstituteFC001066European Union
Wellcome Trust106252/Z/14/ZEuropean Union
European Research Council (ERC)669424-CHROMOREPEuropean Union
European Research Council (ERC)820102European Union
CitationJournal: Nature / Year: 2022
Title: Mechanism of replication origin melting nucleated by CMG helicase assembly.
Authors: Jacob S Lewis / Marta H Gross / Joana Sousa / Sarah S Henrikus / Julia F Greiwe / Andrea Nans / John F X Diffley / Alessandro Costa /
Abstract: The activation of eukaryotic origins of replication occurs in temporally separated steps to ensure that chromosomes are copied only once per cell cycle. First, the MCM helicase is loaded onto duplex ...The activation of eukaryotic origins of replication occurs in temporally separated steps to ensure that chromosomes are copied only once per cell cycle. First, the MCM helicase is loaded onto duplex DNA as an inactive double hexamer. Activation occurs after the recruitment of a set of firing factors that assemble two Cdc45-MCM-GINS (CMG) holo-helicases. CMG formation leads to the underwinding of DNA on the path to the establishment of the replication fork, but whether DNA becomes melted at this stage is unknown. Here we use cryo-electron microscopy to image ATP-dependent CMG assembly on a chromatinized origin, reconstituted in vitro with purified yeast proteins. We find that CMG formation disrupts the double hexamer interface and thereby exposes duplex DNA in between the two CMGs. The two helicases remain tethered, which gives rise to a splayed dimer, with implications for origin activation and replisome integrity. Inside each MCM ring, the double helix becomes untwisted and base pairing is broken. This comes as the result of ATP-triggered conformational changes in MCM that involve DNA stretching and protein-mediated stabilization of three orphan bases. Mcm2 pore-loop residues that engage DNA in our structure are dispensable for double hexamer loading and CMG formation, but are essential to untwist the DNA and promote replication. Our results explain how ATP binding nucleates origin DNA melting by the CMG and maintains replisome stability at initiation.
History
DepositionDec 14, 2021-
Header (metadata) releaseJun 15, 2022-
Map releaseJun 15, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13988.png

Downloads & links

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Map

FileDownload / File: emd_13988.map.gz / Format: CCP4 / Size: 824 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationcryoSPARC trans CMGE dimer
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.3 Å/pix.
x 600 pix.
= 777.6 Å		1.3 Å/pix.
x 600 pix.
= 777.6 Å		1.3 Å/pix.
x 600 pix.
= 777.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.296 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.23
Minimum - Maximum-0.70822626 - 1.2658054
Average (Standard dev.)0.0019956823 (±0.031100279)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions600600600
Spacing600600600
CellA=B=C: 777.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: cryoSPARC trans CMGE dimer half map 1

Fileemd_13988_half_map_1.map
AnnotationcryoSPARC trans CMGE dimer half map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: cryoSPARC trans CMGE dimer half map 2

Fileemd_13988_half_map_2.map
AnnotationcryoSPARC trans CMGE dimer half map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ternary complex of the CMG helicase melting origin DNA

EntireName: Ternary complex of the CMG helicase melting origin DNA
Components
  • Complex: Ternary complex of the CMG helicase melting origin DNA

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Supramolecule #1: Ternary complex of the CMG helicase melting origin DNA

SupramoleculeName: Ternary complex of the CMG helicase melting origin DNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#15
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 / Instrument: FEI VITROBOT MARK IV
Detailsfour microlitres of sample was applied on a grid and incubated for 2 min at room temperature before blotting with filter paper for 5.5 s and plunge-freezing in liquid ethane.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 4.4 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 2 / Number real images: 65286 / Average exposure time: 10.0 sec. / Average electron dose: 1.6 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 927109
Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 8.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Number images used: 49766

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