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- EMDB-6465: The Architecture of a Eukaryotic Replisome -

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Basic information

Entry
Database: EMDB / ID: EMD-6465
TitleThe Architecture of a Eukaryotic Replisome
Map dataYeast CMGE complex
Sample
  • Sample: Saccharomyces cerevisiae CMGE complex
  • Protein or peptide: CMG complex
  • Protein or peptide: Polymerase Epsilon
KeywordsEukaryotic replisome / CMGE / Electron microscopy
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / negative staining / Resolution: 16.0 Å
AuthorsSun J / Shi Y / Georgescu RE / Yuan Z / Chait B / Li H / O'Donnell ME
CitationJournal: Nat Struct Mol Biol / Year: 2015
Title: The architecture of a eukaryotic replisome.
Authors: Jingchuan Sun / Yi Shi / Roxana E Georgescu / Zuanning Yuan / Brian T Chait / Huilin Li / Michael E O'Donnell /
Abstract: At the eukaryotic DNA replication fork, it is widely believed that the Cdc45-Mcm2-7-GINS (CMG) helicase is positioned in front to unwind DNA and that DNA polymerases trail behind the helicase. Here ...At the eukaryotic DNA replication fork, it is widely believed that the Cdc45-Mcm2-7-GINS (CMG) helicase is positioned in front to unwind DNA and that DNA polymerases trail behind the helicase. Here we used single-particle EM to directly image a Saccharomyces cerevisiae replisome. Contrary to expectations, the leading strand Pol ɛ is positioned ahead of CMG helicase, whereas Ctf4 and the lagging-strand polymerase (Pol) α-primase are behind the helicase. This unexpected architecture indicates that the leading-strand DNA travels a long distance before reaching Pol ɛ, first threading through the Mcm2-7 ring and then making a U-turn at the bottom and reaching Pol ɛ at the top of CMG. Our work reveals an unexpected configuration of the eukaryotic replisome, suggests possible reasons for this architecture and provides a basis for further structural and biochemical replisome studies.
History
DepositionSep 10, 2015-
Header (metadata) releaseOct 14, 2015-
Map releaseNov 4, 2015-
UpdateDec 16, 2015-
Current statusDec 16, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6465.jpg

Downloads & links

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Map

FileDownload / File: emd_6465.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationYeast CMGE complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.12 Å/pix.
x 256 pix.
= 542.72 Å		2.12 Å/pix.
x 256 pix.
= 542.72 Å		2.12 Å/pix.
x 256 pix.
= 542.72 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.12 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 0.408 / Movie #1: 0.5
Minimum - Maximum-0.43355834 - 0.9493196
Average (Standard dev.)0.0007616 (±0.06425492)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-64-64
Dimensions256256256
Spacing256256256
CellA=B=C: 542.72 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.122.122.12
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z542.720542.720542.720
α/β/γ90.00090.00090.000
start NX/NY/NZ-190-190-189
NX/NY/NZ380380380
MAP C/R/S123
start NC/NR/NS-64-64-64
NC/NR/NS256256256
D min/max/mean-0.4340.9490.001

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Supplemental data

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Sample components

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Entire : Saccharomyces cerevisiae CMGE complex

EntireName: Saccharomyces cerevisiae CMGE complex
Components
  • Sample: Saccharomyces cerevisiae CMGE complex
  • Protein or peptide: CMG complex
  • Protein or peptide: Polymerase Epsilon

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Supramolecule #1000: Saccharomyces cerevisiae CMGE complex

SupramoleculeName: Saccharomyces cerevisiae CMGE complex / type: sample / ID: 1000 / Number unique components: 2
Molecular weightTheoretical: 1.2 MDa

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Macromolecule #1: CMG complex

MacromoleculeName: CMG complex / type: protein_or_peptide / ID: 1 / Name.synonym: CMG / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Yeast
Molecular weightTheoretical: 700 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)

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Macromolecule #2: Polymerase Epsilon

MacromoleculeName: Polymerase Epsilon / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Yeast
Molecular weightTheoretical: 500 KDa
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

Concentration0.5 mg/mL
BufferDetails: 20 mM Tris-acetate, 40 mM potassium glutamate, 2 mM DTT, 0.1 mM EDTA
StainingType: NEGATIVE
Details: Grids were stained with 1% w/v uranyl acetate for 1 minute.
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeJEOL 2010F
DateSep 10, 2014
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 320 / Average electron dose: 20 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal magnification: 50000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN

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Image processing

DetailsParticles were selected in EMAN 2.1 using SWAM.
CTF correctionDetails: CTFFIND3
Final reconstructionResolution.type: BY AUTHOR / Resolution: 16.0 Å / Resolution method: OTHER / Software - Name: Relion_1.3 / Number images used: 18721

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