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- EMDB-3642: Full-length complex of CMG helicase with polymerase epsilon -

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Basic information

Entry
Database: EMDB / ID: 3642
TitleFull-length complex of CMG helicase with polymerase epsilon
Map data
SampleComplex of CMG helicase with full length polymerase epsilon
SourceSaccharomyces cerevisiae / yeast / /
Methodsingle particle reconstruction, at 24.4 Å resolution
AuthorsZhou JC / Janska A / Goswami P / Renault L / Abid Ali F / Kotecha A / Diffley JFX / Costa A
CitationJournal: Proc. Natl. Acad. Sci. U.S.A. / Year: 2017
Title: CMG-Pol epsilon dynamics suggests a mechanism for the establishment of leading-strand synthesis in the eukaryotic replisome.
Authors: Jin Chuan Zhou / Agnieszka Janska / Panchali Goswami / Ludovic Renault / Ferdos Abid Ali / Abhay Kotecha / John F X Diffley / Alessandro Costa
Abstract: The replisome unwinds and synthesizes DNA for genome duplication. In eukaryotes, the Cdc45-MCM-GINS (CMG) helicase and the leading-strand polymerase, Pol epsilon, form a stable assembly. The ...The replisome unwinds and synthesizes DNA for genome duplication. In eukaryotes, the Cdc45-MCM-GINS (CMG) helicase and the leading-strand polymerase, Pol epsilon, form a stable assembly. The mechanism for coupling DNA unwinding with synthesis is starting to be elucidated, however the architecture and dynamics of the replication fork remain only partially understood, preventing a molecular understanding of chromosome replication. To address this issue, we conducted a systematic single-particle EM study on multiple permutations of the reconstituted CMG-Pol epsilon assembly. Pol epsilon contains two flexibly tethered lobes. The noncatalytic lobe is anchored to the motor of the helicase, whereas the polymerization domain extends toward the side of the helicase. We observe two alternate configurations of the DNA synthesis domain in the CMG-bound Pol epsilon. We propose that this conformational switch might control DNA template engagement and release, modulating replisome progression.
DateDeposition: Mar 20, 2017 / Header (metadata) release: Mar 29, 2017 / Map release: Apr 12, 2017 / Last update: Apr 26, 2017

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0321
  • Imaged by UCSF CHIMERA
  • Download
  • Surface view colored by radius
  • Surface level: 0.0321
  • Imaged by UCSF CHIMERA
  • Download
3D viewer
Supplemental images

Downloads & links

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Map

Fileemd_3642.map.gz (map file in CCP4 format, 8389 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
128 pix
3.65 Å/pix.
= 467.2 Å
128 pix
3.65 Å/pix.
= 467.2 Å
128 pix
3.65 Å/pix.
= 467.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider package.

Voxel sizeX=Y=Z: 3.65 Å
Density
Contour Level:0.0321 (by author), 0.0321 (movie #1):
Minimum - Maximum-0.04973585 - 0.11394457
Average (Standard dev.)-6.4426036E-5 (0.008389878)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions128128128
Origin000
Limit127127127
Spacing128128128
CellA=B=C: 467.2 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.653.653.65
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z467.200467.200467.200
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.0500.114-0.000

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Supplemental data

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Sample components

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Entire Complex of CMG helicase with full length polymerase epsilon

EntireName: Complex of CMG helicase with full length polymerase epsilon
Number of components: 1

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Component #1: protein, Complex of CMG helicase with full length polymerase epsilon

ProteinName: Complex of CMG helicase with full length polymerase epsilon
Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae / yeast / /
Source (engineered)Expression System: Saccharomyces cerevisiae / yeast / /

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Experimental details

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Sample preparation

Specimen stateparticle
Sample solutionpH: 7.6
VitrificationCryogen name: NONE

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Electron microscopy imaging

ImagingMicroscope: JEOL 2100
Electron gunElectron source: LAB6 / Accelerating voltage: 120 kV / Electron dose: 35 e/Å2 / Illumination mode: SPOT SCAN
LensImaging mode: OTHER
Specimen HolderModel: OTHER
CameraDetector: GATAN ULTRASCAN 4000 (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 24697
3D reconstructionResolution: 24.4 Å / Resolution method: FSC 0.143 CUT-OFF

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