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- EMDB-51399: OCCM maturation intermediate stalled with an Arginine Finger muta... -

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Basic information

Entry
Database: EMDB / ID: EMD-51399
TitleOCCM maturation intermediate stalled with an Arginine Finger mutation in Mcm5: Conformer 2. ORC local refinement.
Map dataORC local refinement, unmodified map
Sample
  • Complex: ORC-Cdt1-MCM complex
KeywordsAAA+ ATPase / DNA translocase / ATPase switch / DNA replication / REPLICATION
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsButryn A / Costa A
Funding supportEuropean Union, United Kingdom, 2 items
OrganizationGrant numberCountry
European Research Council (ERC)820102European Union
The Francis Crick InstituteCC2009 United Kingdom
CitationJournal: Nat Commun / Year: 2025
Title: Unidirectional MCM translocation away from ORC drives origin licensing.
Authors: Agata Butryn / Julia F Greiwe / Alessandro Costa /
Abstract: The MCM motor of the eukaryotic replicative helicase is loaded as a double hexamer onto DNA by the Origin Recognition Complex (ORC), Cdc6, and Cdt1. ATP binding supports formation of the ORC-Cdc6- ...The MCM motor of the eukaryotic replicative helicase is loaded as a double hexamer onto DNA by the Origin Recognition Complex (ORC), Cdc6, and Cdt1. ATP binding supports formation of the ORC-Cdc6-Cdt1-MCM (OCCM) helicase-recruitment complex where ORC-Cdc6 and one MCM hexamer form two juxtaposed rings around duplex DNA. ATP hydrolysis by MCM completes MCM loading but the mechanism is unknown. Here, we used cryo-EM to characterise helicase loading with ATPase-dead Arginine Finger variants of the six MCM subunits. We report the structure of two MCM complexes with different DNA grips, stalled as they mature to loaded MCM. The Mcm2 Arginine Finger-variant stabilises DNA binding by Mcm2 away from ORC/Cdc6. The Arginine Finger-variant of the neighbouring Mcm5 subunit stabilises DNA engagement by Mcm5 downstream of the Mcm2 binding site. Cdc6 and Orc1 progressively disengage from ORC as MCM translocates along DNA. We observe that duplex DNA translocation by MCM involves a set of leading-strand contacts by the pre-sensor 1 ATPase hairpins and lagging-strand contacts by the helix-2-insert hairpins. Mutating any of the MCM residues involved impairs high-salt resistant DNA binding in vitro and double-hexamer formation assessed by electron microscopy. Thus, ATPase-powered duplex DNA translocation away from ORC underlies MCM loading.
History
DepositionAug 22, 2024-
Header (metadata) releaseMar 5, 2025-
Map releaseMar 5, 2025-
UpdateMar 5, 2025-
Current statusMar 5, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_51399.png

Downloads & links

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Map

FileDownload / File: emd_51399.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationORC local refinement, unmodified map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.95 Å/pix.
x 440 pix.
= 418. Å		0.95 Å/pix.
x 440 pix.
= 418. Å		0.95 Å/pix.
x 440 pix.
= 418. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.95 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.14
Minimum - Maximum-0.5048532 - 0.8917211
Average (Standard dev.)0.0018167304 (±0.021892902)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 418.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: ORC local refinement, density-modified with DeepEMhancer

Fileemd_51399_additional_1.map
AnnotationORC local refinement, density-modified with DeepEMhancer
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-map

Fileemd_51399_half_map_1.map
Annotationhalf-map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-map

Fileemd_51399_half_map_2.map
Annotationhalf-map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : ORC-Cdt1-MCM complex

EntireName: ORC-Cdt1-MCM complex
Components
  • Complex: ORC-Cdt1-MCM complex

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Supramolecule #1: ORC-Cdt1-MCM complex

SupramoleculeName: ORC-Cdt1-MCM complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#14
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 1.1 MDa

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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

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 30.34 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.7 µm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 151030
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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