+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-2772 | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Title | CMG helicase bound to DNA and ATPgS | |||||||||
Map data | Reconstruction of the recombinant Drosophila CMG helicase bound to ATPgS and DNA | |||||||||
Sample |
| |||||||||
Keywords | Helicase / AAA+ ATPase / DNA replication | |||||||||
Function / homology | Function and homology information Unwinding of DNA / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / DNA endoreduplication / Activation of ATR in response to replication stress / Activation of the pre-replicative complex / eggshell chorion gene amplification / Orc1 removal from chromatin / DNA amplification / DNA strand elongation involved in mitotic DNA replication ...Unwinding of DNA / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / DNA endoreduplication / Activation of ATR in response to replication stress / Activation of the pre-replicative complex / eggshell chorion gene amplification / Orc1 removal from chromatin / DNA amplification / DNA strand elongation involved in mitotic DNA replication / GINS complex / mitotic DNA replication preinitiation complex assembly / resolution of meiotic recombination intermediates / premeiotic DNA replication / mitotic DNA replication / CMG complex / DNA replication preinitiation complex / MCM complex / double-strand break repair via break-induced replication / mitotic DNA replication initiation / chromosome condensation / DNA duplex unwinding / DNA strand elongation involved in DNA replication / DNA unwinding involved in DNA replication / DNA replication origin binding / DNA replication initiation / DNA helicase activity / mitotic spindle organization / regulation of DNA-templated transcription elongation / meiotic cell cycle / helicase activity / single-stranded DNA binding / mitotic cell cycle / DNA helicase / DNA replication / cell division / chromatin binding / ATP hydrolysis activity / ATP binding / nucleus / metal ion binding / cytosol / cytoplasm Similarity search - Function | |||||||||
Biological species | Drosophila melanogaster (fruit fly) / unidentified (others) | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 17.8 Å | |||||||||
Authors | Costa A / Renault L / Swuec P / Petojevic T / Pesavento JJ / Ilves I / MacLellan-Gibson K / Fleck RA / Botchan MR / Berger JM | |||||||||
Citation | Journal: Elife / Year: 2014 Title: DNA binding polarity, dimerization, and ATPase ring remodeling in the CMG helicase of the eukaryotic replisome. Authors: Alessandro Costa / Ludovic Renault / Paolo Swuec / Tatjana Petojevic / James J Pesavento / Ivar Ilves / Kirsty MacLellan-Gibson / Roland A Fleck / Michael R Botchan / James M Berger / Abstract: The Cdc45/Mcm2-7/GINS (CMG) helicase separates DNA strands during replication in eukaryotes. How the CMG is assembled and engages DNA substrates remains unclear. Using electron microscopy, we have ...The Cdc45/Mcm2-7/GINS (CMG) helicase separates DNA strands during replication in eukaryotes. How the CMG is assembled and engages DNA substrates remains unclear. Using electron microscopy, we have determined the structure of the CMG in the presence of ATPγS and a DNA duplex bearing a 3' single-stranded tail. The structure shows that the MCM subunits of the CMG bind preferentially to single-stranded DNA, establishes the polarity by which DNA enters into the Mcm2-7 pore, and explains how Cdc45 helps prevent DNA from dissociating from the helicase. The Mcm2-7 subcomplex forms a cracked-ring, right-handed spiral when DNA and nucleotide are bound, revealing unexpected congruencies between the CMG and both bacterial DnaB helicases and the AAA+ motor of the eukaryotic proteasome. The existence of a subpopulation of dimeric CMGs establishes the subunit register of Mcm2-7 double hexamers and together with the spiral form highlights how Mcm2-7 transitions through different conformational and assembly states as it matures into a functional helicase. | |||||||||
History |
|
-Structure visualization
Movie |
Movie viewer |
---|---|
Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_2772.map.gz | 15.9 MB | EMDB map data format | |
---|---|---|---|---|
Header (meta data) | emd-2772-v30.xml emd-2772.xml | 22.7 KB 22.7 KB | Display Display | EMDB header |
Images | EMD-2772-deposition_image.png | 79 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-2772 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-2772 | HTTPS FTP |
-Validation report
Summary document | emd_2772_validation.pdf.gz | 228.3 KB | Display | EMDB validaton report |
---|---|---|---|---|
Full document | emd_2772_full_validation.pdf.gz | 227.4 KB | Display | |
Data in XML | emd_2772_validation.xml.gz | 5.8 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2772 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2772 | HTTPS FTP |
-Related structure data
Similar structure data |
---|
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
---|---|
Related items in Molecule of the Month |
-Map
File | Download / File: emd_2772.map.gz / Format: CCP4 / Size: 17.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | Reconstruction of the recombinant Drosophila CMG helicase bound to ATPgS and DNA | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.14 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
|
-Supplemental data
-Sample components
+Entire : Drosophila melanogaster Cdc45-Mcm2-7-GINS complex bound to ATPgS ...
+Supramolecule #1000: Drosophila melanogaster Cdc45-Mcm2-7-GINS complex bound to ATPgS ...
+Macromolecule #1: Mcm2
+Macromolecule #2: Mcm3
+Macromolecule #3: Mcm4
+Macromolecule #4: Mcm5
+Macromolecule #5: Mcm6
+Macromolecule #6: Mcm7
+Macromolecule #7: Cdc45
+Macromolecule #8: Psf1
+Macromolecule #9: Psf2
+Macromolecule #10: Psf3
+Macromolecule #11: Sld5
+Macromolecule #12: LEAD60
+Macromolecule #13: 3BTNLAG20
-Experimental details
-Structure determination
Method | negative staining |
---|---|
Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.02 mg/mL |
---|---|
Buffer | pH: 7.6 Details: 25 mM HEPES , 50 mM sodium acetate, 10 mM magnesium acetate, 0.1 mM ATPgS, 1 mM DTT |
Staining | Type: NEGATIVE Details: The grid with adsorbed protein was floated for 10 seconds on four consecutive 75 microliter drops of 2% w/v uranyl formate solution. |
Grid | Details: 400 mesh copper grids with thin carbon support. |
Vitrification | Cryogen name: NONE / Instrument: OTHER Details: The sample stained with a fresh 2% (wt/vol) uranyl formate solution. |
-Electron microscopy
Microscope | JEOL 2100 |
---|---|
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 60,000 times magnification |
Date | May 8, 2013 |
Image recording | Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 579 / Average electron dose: 35 e/Å2 / Bits/pixel: 32 |
Electron beam | Acceleration voltage: 200 kV / Electron source: LAB6 |
Electron optics | Calibrated magnification: 70000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 50000 |
Sample stage | Specimen holder: single tilt room temperature / Specimen holder model: JEOL |