|Entry||Database: EMDB / ID: EMD-6535|
|Title||Structure of the eukaryotic replicative CMG helicase and pumpjack motion|
|Sample||Saccharomyces cerevisiae CMG complex:|
|Keywords||CMG helicase / Cryo-EM|
|Function / homology|
Function and homology information
MCM complex binding / MCM core complex / nuclear DNA replication / CMG complex / GINS complex / DNA strand elongation involved in mitotic DNA replication / mitotic DNA replication preinitiation complex assembly / negative regulation of chromatin silencing at telomere / mitotic DNA replication / regulation of chromatin silencing at telomere ...MCM complex binding / MCM core complex / nuclear DNA replication / CMG complex / GINS complex / DNA strand elongation involved in mitotic DNA replication / mitotic DNA replication preinitiation complex assembly / negative regulation of chromatin silencing at telomere / mitotic DNA replication / regulation of chromatin silencing at telomere / establishment of chromatin silencing / negative regulation of DNA helicase activity / regulation of DNA-dependent DNA replication initiation / MCM complex / nuclear pre-replicative complex / DNA replication preinitiation complex / mitotic DNA replication initiation / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / single-stranded DNA helicase activity / double-strand break repair via break-induced replication / replication fork protection complex / nuclear replication fork / 3'-5' DNA helicase activity / DNA strand elongation involved in DNA replication / chromatin silencing at silent mating-type cassette / DNA duplex unwinding / DNA unwinding involved in DNA replication / DNA replication origin binding / chromatin silencing at telomere / DNA replication initiation / positive regulation of G1/S transition of mitotic cell cycle / DNA helicase / DNA helicase activity / chromosome, telomeric region / single-stranded DNA binding / DNA-dependent DNA replication / nuclear chromosome, telomeric region / cellular response to DNA damage stimulus / chromatin binding / nucleoplasm / ATP binding / metal ion binding / nucleus / cytoplasm
GINS complex, subunit Psf3 / DNA replication licensing factor Mcm6 / Nucleic acid-binding, OB-fold / MCM domain / AAA+ ATPase domain / CDC45 family / GINS complex, subunit Psf1 / GINS subunit, domain A / DNA replication complex GINS protein Psf2 / DNA replication licensing factor Mcm2 ...GINS complex, subunit Psf3 / DNA replication licensing factor Mcm6 / Nucleic acid-binding, OB-fold / MCM domain / AAA+ ATPase domain / CDC45 family / GINS complex, subunit Psf1 / GINS subunit, domain A / DNA replication complex GINS protein Psf2 / DNA replication licensing factor Mcm2 / DNA replication licensing factor Mcm3 / Mini-chromosome maintenance complex protein 4 / Mini-chromosome maintenance, conserved site / DNA replication licensing factor Mcm5 / P-loop containing nucleoside triphosphate hydrolase / GINS complex subunit Sld5 / MCM, AAA-lid domain / Mcm6, C-terminal winged-helix domain / MCM N-terminal domain / GINS complex, subunit Psf3 superfamily / GINS complex protein Sld5, alpha-helical domain / GINS, helical bundle-like domain superfamily / MCM OB domain / DNA replication complex GINS protein SLD5, C-terminal / Mini-chromosome maintenance protein / DNA replication licensing factor Mcm7
DNA replication licensing factor MCM3 / DNA replication licensing factor MCM2 / Minichromosome maintenance protein 5 / DNA replication licensing factor MCM4 / DNA replication licensing factor MCM7 / DNA replication complex GINS protein PSF2 / DNA replication licensing factor MCM6 / DNA replication complex GINS protein SLD5 / Cell division control protein 45 / DNA replication complex GINS protein PSF3 / DNA replication complex GINS protein PSF1
|Biological species||Saccharomyces cerevisiae (baker's yeast)|
|Method||single particle reconstruction / cryo EM / Resolution: 4.7 Å|
|Authors||Yuan Z / Bai L / Sun J / Georgescu RE / Liu J / O'Donnell ME / Li H|
|Citation||Journal: Nat. Struct. Mol. Biol. / Year: 2016|
Title: Structure of the eukaryotic replicative CMG helicase suggests a pumpjack motion for translocation.
Authors: Zuanning Yuan / Lin Bai / Jingchuan Sun / Roxana Georgescu / Jun Liu / Michael E O'Donnell / Huilin Li /
Abstract: The CMG helicase is composed of Cdc45, Mcm2-7 and GINS. Here we report the structure of the Saccharomyces cerevisiae CMG, determined by cryo-EM at a resolution of 3.7-4.8 Å. The structure reveals ...The CMG helicase is composed of Cdc45, Mcm2-7 and GINS. Here we report the structure of the Saccharomyces cerevisiae CMG, determined by cryo-EM at a resolution of 3.7-4.8 Å. The structure reveals that GINS and Cdc45 scaffold the N tier of the helicase while enabling motion of the AAA+ C tier. CMG exists in two alternating conformations, compact and extended, thus suggesting that the helicase moves like an inchworm. The N-terminal regions of Mcm2-7, braced by Cdc45-GINS, form a rigid platform upon which the AAA+ C domains make longitudinal motions, nodding up and down like an oil-rig pumpjack attached to a stable platform. The Mcm ring is remodeled in CMG relative to the inactive Mcm2-7 double hexamer. The Mcm5 winged-helix domain is inserted into the central channel, thus blocking entry of double-stranded DNA and supporting a steric-exclusion DNA-unwinding model.
|Validation Report||PDB-ID: 3jc5|
SummaryFull reportAbout validation report
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_6535.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.01 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Entire Saccharomyces cerevisiae CMG complex
|Entire||Name: Saccharomyces cerevisiae CMG complex / Number of components: 1|
|Mass||Theoretical: 700 kDa|
-Component #1: protein, CMG complex
|Protein||Name: CMG complex / a.k.a: CMG / Recombinant expression: Yes / Number of Copies: 1|
|Mass||Theoretical: 700 kDa|
|Source||Species: Saccharomyces cerevisiae (baker's yeast)|
|Source (engineered)||Expression System: Saccharomyces cerevisiae (baker's yeast)|
|Specimen||Specimen state: Particle / Method: cryo EM|
|Sample solution||Specimen conc.: 0.6 mg/mL|
Buffer solution: 20 mM Tris acetate, 40 mM potassium glutamate, 2 mM DTT, 0.1 mM EDTA
|Support film||400 mesh holey carbon C-flat grid, glow-discharged in air|
|Vitrification||Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Method: Blot for 3 seconds before plunging|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Imaging||Microscope: FEI TITAN KRIOS / Date: Aug 1, 2015|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 50 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 29000 X (nominal), 49505 X (calibrated) / Imaging mode: BRIGHT FIELD / Defocus: 1500 - 3500 nm|
|Specimen Holder||Model: FEI TITAN KRIOS AUTOGRID HOLDER|
|Camera||Detector: GATAN K2 SUMMIT (4k x 4k)|
|Image acquisition||Number of digital images: 8000|
|Processing||Method: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 178530 |
Details: All steps, including automatic particle picking, 2D classification, 3D classification, and 3D refinement were performed in Relion 1.4.
|3D reconstruction||Software: Relion_1.4 / CTF correction: CTFFIND4 / Resolution: 4.7 Å / Resolution method: FSC 0.143, gold-standard|
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