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- EMDB-4788: D. melanogaster CMG-DNA with ATP, State 2B -

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Basic information

Entry
Database: EMDB / ID: EMD-4788
TitleD. melanogaster CMG-DNA with ATP, State 2B
Map data
SampleCMG helicase bound to forked DNA in the presence of ATP, State 2B
  • CMG helicase
  • DNA
  • (nucleic-acidNucleic acid) x 2
  • CDC45LCDC45-related protein
  • IP07275p
  • Probable DNA replication complex GINS protein PSF2
  • AT18545p
  • DNA replication complex GINS protein SLD5
  • (DNA replication licensing factor ...) x 6
  • (ligand) x 2
Function / homology
Function and homology information


Assembly of the pre-replicative complex / Activation of ATR in response to replication stress / Activation of the pre-replicative complex / Switching of origins to a post-replicative state / Unwinding of DNA / DNA endoreduplication / DNA amplification / eggshell chorion gene amplification / GINS complex / DNA strand elongation involved in mitotic DNA replication ...Assembly of the pre-replicative complex / Activation of ATR in response to replication stress / Activation of the pre-replicative complex / Switching of origins to a post-replicative state / Unwinding of DNA / DNA endoreduplication / DNA amplification / eggshell chorion gene amplification / GINS complex / DNA strand elongation involved in mitotic DNA replication / mitotic DNA replication preinitiation complex assembly / regulation of chromatin silencing at telomere / mitotic DNA replication / negative regulation of DNA helicase activity / 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 / resolution of meiotic recombination intermediates / double-strand break repair via break-induced replication / regulation of DNA-templated transcription, elongation / replication fork protection complex / DNA strand elongation involved in DNA replication / chromosome condensation / 3'-5' DNA helicase activity / DNA duplex unwinding / DNA unwinding involved in DNA replication / DNA replication origin binding / DNA replication initiation / positive regulation of G1/S transition of mitotic cell cycle / DNA helicase / mitotic spindle organization / DNA helicase activity / meiotic cell cycle / DNA-dependent DNA replication / single-stranded DNA binding / mitotic cell cycle / DNA replication / cell cycle / cell division / chromatin binding / DNA binding / ATP binding / metal ion binding / nucleus / cytosol / cytoplasm
DNA replication licensing factor Mcm2 / DNA replication licensing factor Mcm7 / CDC45-like protein / MCM domain / AAA+ ATPase domain / CDC45 family / GINS complex, subunit Psf1 / DNA replication complex GINS protein Psf2 / MCM P-loop domain / DNA replication licensing factor Mcm3 ...DNA replication licensing factor Mcm2 / DNA replication licensing factor Mcm7 / CDC45-like protein / MCM domain / AAA+ ATPase domain / CDC45 family / GINS complex, subunit Psf1 / DNA replication complex GINS protein Psf2 / MCM P-loop domain / DNA replication licensing factor Mcm3 / Mini-chromosome maintenance complex protein 4 / DNA replication licensing factor Mcm5 / DNA replication licensing factor Mcm6 / GINS complex subunit Sld5 / MCM, AAA-lid domain / GINS complex, subunit Psf3 / Nucleic acid-binding, OB-fold / Mini-chromosome maintenance, conserved site / GINS subunit, domain A / P-loop containing nucleoside triphosphate hydrolase / MCM N-terminal domain / Mini-chromosome maintenance protein / DNA replication complex GINS protein SLD5, C-terminal / MCM OB domain / GINS, helical bundle-like domain superfamily / GINS complex, subunit Psf3 superfamily / GINS complex protein Sld5, alpha-helical domain / GINS complex protein / Mini-chromosome maintenance protein 2 / Mcm6, C-terminal winged-helix domain / MCM family domain profile. / MCM family signature. / MCM6 C-terminal winged-helix domain / MCM AAA-lid domain / MCM OB domain / DNA replication complex GINS protein SLD5 C-terminus / MCM N-terminal domain
Probable DNA replication complex GINS protein PSF2 / DNA replication licensing factor Mcm7 / AT18545p / IP07275p / DNA replication licensing factor MCM4 / DNA replication licensing factor Mcm5 / DNA replication complex GINS protein SLD5 / DNA replication licensing factor Mcm6 / DNA replication licensing factor Mcm2 / CDC45L / DNA replication licensing factor Mcm3
Biological speciesDrosophila melanogaster (fruit fly)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.46 Å
AuthorsEickhoff P / Martino F / Locke J / Nans A / Costa A
CitationJournal: Cell Rep / Year: 2019
Title: Molecular Basis for ATP-Hydrolysis-Driven DNA Translocation by the CMG Helicase of the Eukaryotic Replisome.
Authors: Patrik Eickhoff / Hazal B Kose / Fabrizio Martino / Tatjana Petojevic / Ferdos Abid Ali / Julia Locke / Nele Tamberg / Andrea Nans / James M Berger / Michael R Botchan / Hasan Yardimci / Alessandro Costa /
Abstract: In the eukaryotic replisome, DNA unwinding by the Cdc45-MCM-Go-Ichi-Ni-San (GINS) (CMG) helicase requires a hexameric ring-shaped ATPase named minichromosome maintenance (MCM), which spools ...In the eukaryotic replisome, DNA unwinding by the Cdc45-MCM-Go-Ichi-Ni-San (GINS) (CMG) helicase requires a hexameric ring-shaped ATPase named minichromosome maintenance (MCM), which spools single-stranded DNA through its central channel. Not all six ATPase sites are required for unwinding; however, the helicase mechanism is unknown. We imaged ATP-hydrolysis-driven translocation of the CMG using cryo-electron microscopy (cryo-EM) and found that the six MCM subunits engage DNA using four neighboring protomers at a time, with ATP binding promoting DNA engagement. Morphing between different helicase states leads us to suggest a non-symmetric hand-over-hand rotary mechanism, explaining the asymmetric requirements of ATPase function around the MCM ring of the CMG. By imaging of a higher-order replisome assembly, we find that the Mrc1-Csm3-Tof1 fork-stabilization complex strengthens the interaction between parental duplex DNA and the CMG at the fork, which might support the coupling between DNA translocation and fork unwinding.
Validation ReportPDB-ID: 6raz

SummaryFull reportAbout validation report
History
DepositionApr 8, 2019-
Header (metadata) releaseSep 18, 2019-
Map releaseSep 18, 2019-
UpdateSep 18, 2019-
Current statusSep 18, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0045
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0045
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6raz
  • Surface level: 0.0045
  • 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_4788.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 384 pix.
= 414.72 Å
1.08 Å/pix.
x 384 pix.
= 414.72 Å
1.08 Å/pix.
x 384 pix.
= 414.72 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.0045 / Movie #1: 0.0045
Minimum - Maximum-0.0199342 - 0.03066142
Average (Standard dev.)0.00001888085 (±0.00094299926)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 414.72003 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z414.720414.720414.720
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-0.0270.0390.000

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Supplemental data

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Sample components

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Entire CMG helicase bound to forked DNA in the presence of ATP, State 2B

EntireName: CMG helicase bound to forked DNA in the presence of ATP, State 2B
Number of components: 18

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Component #1: protein, CMG helicase bound to forked DNA in the presence of ATP,...

ProteinName: CMG helicase bound to forked DNA in the presence of ATP, State 2B
Recombinant expression: No

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Component #2: protein, CMG helicase

ProteinName: CMG helicase / Recombinant expression: No
SourceSpecies: Drosophila melanogaster (fruit fly)
Source (engineered)Expression System: Trichoplusia ni (cabbage looper)

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Component #3: protein, DNA

ProteinName: DNA / Recombinant expression: No
SourceSpecies: Drosophila melanogaster (fruit fly)
Source (engineered)Expression System: synthetic construct (others)

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Component #4: nucleic-acid, DNA

nucleic acidName: DNA / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DC)(DG)(DT)(DT)(DT)(DT)(DA)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DA)(DA) (DA)
MassTheoretical: 6.389149 kDa
SourceSpecies: Drosophila melanogaster (fruit fly)

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Component #5: nucleic-acid, DNA

nucleic acidName: DNA / Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
GC
MassTheoretical: 0.60543 kDa
SourceSpecies: Drosophila melanogaster (fruit fly)

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Component #6: protein, CDC45L

ProteinName: CDC45LCDC45-related protein / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 65.968789 kDa
SourceSpecies: Drosophila melanogaster (fruit fly)
Source (engineered)Expression System: Trichoplusia ni (cabbage looper)

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Component #7: protein, IP07275p

ProteinName: IP07275p / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 23.333693 kDa
SourceSpecies: Drosophila melanogaster (fruit fly)
Source (engineered)Expression System: Trichoplusia ni (cabbage looper)

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Component #8: protein, Probable DNA replication complex GINS protein PSF2

ProteinName: Probable DNA replication complex GINS protein PSF2 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 23.141781 kDa
SourceSpecies: Drosophila melanogaster (fruit fly)
Source (engineered)Expression System: Trichoplusia ni (cabbage looper)

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Component #9: protein, AT18545p

ProteinName: AT18545p / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 24.829984 kDa
SourceSpecies: Drosophila melanogaster (fruit fly)
Source (engineered)Expression System: Trichoplusia ni (cabbage looper)

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Component #10: protein, DNA replication complex GINS protein SLD5

ProteinName: DNA replication complex GINS protein SLD5 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 26.148234 kDa
SourceSpecies: Drosophila melanogaster (fruit fly)
Source (engineered)Expression System: Trichoplusia ni (cabbage looper)

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Component #11: protein, DNA replication licensing factor Mcm2

ProteinName: DNA replication licensing factor Mcm2 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 100.537438 kDa
SourceSpecies: Drosophila melanogaster (fruit fly)
Source (engineered)Expression System: Trichoplusia ni (cabbage looper)

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Component #12: protein, DNA replication licensing factor Mcm5

ProteinName: DNA replication licensing factor Mcm5 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 82.37532 kDa
SourceSpecies: Drosophila melanogaster (fruit fly)
Source (engineered)Expression System: Trichoplusia ni (cabbage looper)

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Component #13: protein, DNA replication licensing factor Mcm6

ProteinName: DNA replication licensing factor Mcm6 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 92.46782 kDa
SourceSpecies: Drosophila melanogaster (fruit fly)
Source (engineered)Expression System: Trichoplusia ni (cabbage looper)

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Component #14: protein, DNA replication licensing factor Mcm3

ProteinName: DNA replication licensing factor Mcm3 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 91.045164 kDa
SourceSpecies: Drosophila melanogaster (fruit fly)
Source (engineered)Expression System: Trichoplusia ni (cabbage looper)

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Component #15: protein, DNA replication licensing factor MCM4

ProteinName: DNA replication licensing factor MCM4 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 96.735094 kDa
SourceSpecies: Drosophila melanogaster (fruit fly)
Source (engineered)Expression System: Trichoplusia ni (cabbage looper)

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Component #16: protein, DNA replication licensing factor Mcm7

ProteinName: DNA replication licensing factor Mcm7 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 81.399352 kDa
SourceSpecies: Drosophila melanogaster (fruit fly)
Source (engineered)Expression System: Trichoplusia ni (cabbage looper)

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Component #17: ligand, ADENOSINE-5'-DIPHOSPHATE

LigandName: ADENOSINE-5'-DIPHOSPHATE / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 0.427201 kDa

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Component #18: ligand, ADENOSINE-5'-TRIPHOSPHATE

LigandName: ADENOSINE-5'-TRIPHOSPHATE / Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 0.507181 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionpH: 7.6
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 50 e/Å2 / Illumination mode: OTHER
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 61082
3D reconstructionResolution: 4.46 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Output model

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