+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-6465 | |||||||||
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Title | The Architecture of a Eukaryotic Replisome | |||||||||
Map data | Yeast CMGE complex | |||||||||
Sample |
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Keywords | Eukaryotic replisome / CMGE / Electron microscopy | |||||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 16.0 Å | |||||||||
Authors | Sun J / Shi Y / Georgescu RE / Yuan Z / Chait B / Li H / O'Donnell ME | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_6465.map.gz | 58.5 MB | EMDB map data format | |
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Header (meta data) | emd-6465-v30.xml emd-6465.xml | 9.3 KB 9.3 KB | Display Display | EMDB header |
Images | emd_6465.jpg | 21.5 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-6465 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-6465 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_6465.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Yeast CMGE complex | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.12 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Saccharomyces cerevisiae CMGE complex
Entire | Name: Saccharomyces cerevisiae CMGE complex |
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Components |
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-Supramolecule #1000: Saccharomyces cerevisiae CMGE complex
Supramolecule | Name: Saccharomyces cerevisiae CMGE complex / type: sample / ID: 1000 / Number unique components: 2 |
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Molecular weight | Theoretical: 1.2 MDa |
-Macromolecule #1: CMG complex
Macromolecule | Name: CMG complex / type: protein_or_peptide / ID: 1 / Name.synonym: CMG / Number of copies: 1 / Recombinant expression: Yes |
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Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Yeast |
Molecular weight | Theoretical: 700 KDa |
Recombinant expression | Organism: Saccharomyces cerevisiae (brewer's yeast) |
-Macromolecule #2: Polymerase Epsilon
Macromolecule | Name: Polymerase Epsilon / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Recombinant expression: Yes |
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Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Yeast |
Molecular weight | Theoretical: 500 KDa |
Recombinant expression | Organism: Saccharomyces cerevisiae (brewer's yeast) |
-Experimental details
-Structure determination
Method | negative staining |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.5 mg/mL |
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Buffer | Details: 20 mM Tris-acetate, 40 mM potassium glutamate, 2 mM DTT, 0.1 mM EDTA |
Staining | Type: NEGATIVE Details: Grids were stained with 1% w/v uranyl acetate for 1 minute. |
Vitrification | Cryogen name: NONE / Instrument: OTHER |
-Electron microscopy
Microscope | JEOL 2010F |
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Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 50000 |
Sample stage | Specimen holder model: GATAN LIQUID NITROGEN |
Date | Sep 10, 2014 |
Image recording | Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 320 / Average electron dose: 20 e/Å2 |
-Image processing
CTF correction | Details: CTFFIND3 |
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Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 16.0 Å / Resolution method: OTHER / Software - Name: Relion_1.3 / Number images used: 18721 |
Details | Particles were selected in EMAN 2.1 using SWAM. |