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- EMDB-6535: Structure of the eukaryotic replicative CMG helicase and pumpjack... -

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Basic information

Entry
Database: EMDB / ID: EMD-6535
TitleStructure of the eukaryotic replicative CMG helicase and pumpjack motion
Map data
SampleSaccharomyces cerevisiae CMG complex:
CMG complex
KeywordsCMG 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 speciesSaccharomyces cerevisiae (baker's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.7 Å
AuthorsYuan Z / Bai L / Sun J / Georgescu RE / Liu J / O'Donnell ME / Li H
CitationJournal: 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 ReportPDB-ID: 3jc5

SummaryFull reportAbout validation report
History
Current status-Processing site: RCSB / Status: Released
DepositionNov 23, 2015-
Header (metadata) releaseFeb 10, 2016-
Map releaseFeb 10, 2016-
UpdateApr 27, 2016-

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-3jc5
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_6535.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.01 Å/pix.
x 256 pix.
= 258.56 Å
1.01 Å/pix.
x 256 pix.
= 258.56 Å
1.01 Å/pix.
x 256 pix.
= 258.56 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.01 Å
Density
Contour LevelBy EMDB: 0.018 / Movie #1: 0.015
Minimum - Maximum-0.02877966 - 0.06427838
Average (Standard dev.)0.00066107 (±0.00432134)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 258.56 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.011.011.01
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z258.560258.560258.560
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0290.0640.001

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Supplemental data

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Sample components

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Entire Saccharomyces cerevisiae CMG complex

EntireName: Saccharomyces cerevisiae CMG complex / Number of components: 1
MassTheoretical: 700 kDa

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Component #1: protein, CMG complex

ProteinName: CMG complex / a.k.a: CMG / Recombinant expression: Yes / Number of Copies: 1
MassTheoretical: 700 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.6 mg/mL
Buffer solution: 20 mM Tris acetate, 40 mM potassium glutamate, 2 mM DTT, 0.1 mM EDTA
Support film400 mesh holey carbon C-flat grid, glow-discharged in air
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Method: Blot for 3 seconds before plunging

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Date: Aug 1, 2015
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 50 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 29000 X (nominal), 49505 X (calibrated) / Imaging mode: BRIGHT FIELD / Defocus: 1500 - 3500 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 8000

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Image processing

ProcessingMethod: 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 reconstructionSoftware: Relion_1.4 / CTF correction: CTFFIND4 / Resolution: 4.7 Å / Resolution method: FSC 0.143, gold-standard

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Atomic model buiding

Modeling #1Software: Chimera / Refinement protocol: rigid body / Refinement space: REAL
Input PDB model: 2Q9Q
Output model

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