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- EMDB-3679: Full-length complex of S.cerevisiae Cdt1-MCM in apo state. -

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Basic information

Entry
Database: EMDB / ID: EMD-3679
TitleFull-length complex of S.cerevisiae Cdt1-MCM in apo state.
Map data
Sample
  • Complex: Full-length yeast Cdt1-MCM complex in apo state.
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / negative staining / Resolution: 20.4 Å
AuthorsHe J / Frigola J / Kinkelin K / Pye VE / Renault L / Douglas M / Remus D / Cherepanov P / Costa A / Diffley JFX
CitationJournal: Nat Commun / Year: 2017
Title: Cdt1 stabilizes an open MCM ring for helicase loading.
Authors: Jordi Frigola / Jun He / Kerstin Kinkelin / Valerie E Pye / Ludovic Renault / Max E Douglas / Dirk Remus / Peter Cherepanov / Alessandro Costa / John F X Diffley /
Abstract: ORC, Cdc6 and Cdt1 act together to load hexameric MCM, the motor of the eukaryotic replicative helicase, into double hexamers at replication origins. Here we show that Cdt1 interacts with MCM ...ORC, Cdc6 and Cdt1 act together to load hexameric MCM, the motor of the eukaryotic replicative helicase, into double hexamers at replication origins. Here we show that Cdt1 interacts with MCM subunits Mcm2, 4 and 6, which both destabilizes the Mcm2-5 interface and inhibits MCM ATPase activity. Using X-ray crystallography, we show that Cdt1 contains two winged-helix domains in the C-terminal half of the protein and a catalytically inactive dioxygenase-related N-terminal domain, which is important for MCM loading, but not for subsequent replication. We used these structures together with single-particle electron microscopy to generate three-dimensional models of MCM complexes. These show that Cdt1 stabilizes MCM in a left-handed spiral open at the Mcm2-5 gate. We propose that Cdt1 acts as a brace, holding MCM open for DNA entry and bound to ATP until ORC-Cdc6 triggers ATP hydrolysis by MCM, promoting both Cdt1 ejection and MCM ring closure.
History
DepositionApr 20, 2017-
Header (metadata) releaseJun 14, 2017-
Map releaseJul 5, 2017-
UpdateAug 2, 2017-
Current statusAug 2, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.078
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.078
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3679.png

Downloads & links

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Map

FileDownload / File: emd_3679.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 2.7 Å
Density
Contour LevelBy AUTHOR: 0.078 / Movie #1: 0.078
Minimum - Maximum-0.122188404 - 0.26828328
Average (Standard dev.)-0.0008814913 (±0.01966846)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 345.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.72.72.7
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z345.600345.600345.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.1220.268-0.001

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Supplemental data

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Sample components

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Entire : Full-length yeast Cdt1-MCM complex in apo state.

EntireName: Full-length yeast Cdt1-MCM complex in apo state.
Components
  • Complex: Full-length yeast Cdt1-MCM complex in apo state.

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Supramolecule #1: Full-length yeast Cdt1-MCM complex in apo state.

SupramoleculeName: Full-length yeast Cdt1-MCM complex in apo state. / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.6
StainingType: NEGATIVE / Material: Uranyl formate

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Electron microscopy

MicroscopeJEOL 2100
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average electron dose: 20.0 e/Å2

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Image processing

Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 20.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 41013
FSC plot (resolution estimation)

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