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- EMDB-3319: CryoEM structure of the CMG replicative helicase bound to a DNA f... -

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Basic information

Entry
Database: EMDB / ID: EMD-3319
TitleCryoEM structure of the CMG replicative helicase bound to a DNA fork (relaxed state)
Map dataCMG treated with forked DNA substrate in the presence of ATPgS in a relaxed Mcm5-2 AAA+ configuration
Sample
  • Sample: CMG treated with forked DNA substrate and ATPgS
  • Protein or peptide: x 11 types
  • DNA: x 2 types
KeywordsCdc45 / GINS / MCM / CMG / helicase / 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 strand elongation involved in DNA replication / DNA duplex unwinding / 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 / mitotic cell cycle / single-stranded DNA binding / DNA helicase / DNA replication / cell division / chromatin binding / ATP hydrolysis activity / ATP binding / nucleus / metal ion binding / cytosol / cytoplasm
Similarity search - Function
GINS/PriA/YqbF domain / CDC45 family / DNA replication complex GINS protein Psf2 / CDC45 family / DNA replication complex GINS protein Psf2 / CDC45-like protein / GINS complex, subunit Psf3 / GINS complex, subunit Psf1 / GINS complex, subunit Psf3 / GINS complex, subunit Psf3 superfamily ...GINS/PriA/YqbF domain / CDC45 family / DNA replication complex GINS protein Psf2 / CDC45 family / DNA replication complex GINS protein Psf2 / CDC45-like protein / GINS complex, subunit Psf3 / GINS complex, subunit Psf1 / GINS complex, subunit Psf3 / GINS complex, subunit Psf3 superfamily / DNA replication complex GINS protein SLD5, C-terminal / GINS, helical bundle-like domain superfamily / GINS complex protein Sld5, alpha-helical domain / DNA replication complex GINS protein SLD5 C-terminus / GINS complex subunit Sld5 / GINS subunit, domain A / GINS complex subunit Sld5 / GINS subunit, domain A / GINS complex protein helical bundle domain / MCM4, winged helix domain / : / MCM5, C-terminal domain / DNA replication licensing factor Mcm5 / DNA replication licensing factor Mcm5 / DNA replication licensing factor Mcm3 / Mini-chromosome maintenance complex protein 4 / DNA replication licensing factor Mcm6 / DNA replication licensing factor Mcm7 / DNA replication licensing factor Mcm3 / Mini-chromosome maintenance complex protein 4 / DNA replication licensing factor Mcm6 / DNA replication licensing factor Mcm7 / Mcm6, C-terminal winged-helix domain / MCM6 C-terminal winged-helix domain / DNA replication licensing factor Mcm2 / DNA replication licensing factor Mcm2 / Mini-chromosome maintenance protein 2 / Mini-chromosome maintenance, conserved site / MCM family signature. / MCM N-terminal domain / MCM N-terminal domain / MCM OB domain / Mini-chromosome maintenance protein / MCM, AAA-lid domain / MCM P-loop domain / MCM OB domain / MCM AAA-lid domain / MCM family domain profile. / minichromosome maintenance proteins / MCM domain / Winged helix-like DNA-binding domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Nucleic acid-binding, OB-fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
CDC45L / DNA replication licensing factor Mcm2 / DNA replication licensing factor MCM4 / DNA replication licensing factor Mcm6 / DNA replication complex GINS protein SLD5 / DNA replication licensing factor Mcm5 / Probable DNA replication complex GINS protein PSF2 / DNA replication complex GINS protein PSF1 / DNA replication complex GINS protein PSF3 / DNA replication licensing factor Mcm7 / DNA replication licensing factor Mcm3
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.8 Å
AuthorsAbid Ali F / Renault L / Costa A
CitationJournal: Nat Commun / Year: 2016
Title: Cryo-EM structures of the eukaryotic replicative helicase bound to a translocation substrate.
Authors: Ferdos Abid Ali / Ludovic Renault / Julian Gannon / Hailey L Gahlon / Abhay Kotecha / Jin Chuan Zhou / David Rueda / Alessandro Costa /
Abstract: The Cdc45-MCM-GINS (CMG) helicase unwinds DNA during the elongation step of eukaryotic genome duplication and this process depends on the MCM ATPase function. Whether CMG translocation occurs on ...The Cdc45-MCM-GINS (CMG) helicase unwinds DNA during the elongation step of eukaryotic genome duplication and this process depends on the MCM ATPase function. Whether CMG translocation occurs on single- or double-stranded DNA and how ATP hydrolysis drives DNA unwinding remain open questions. Here we use cryo-electron microscopy to describe two subnanometre resolution structures of the CMG helicase trapped on a DNA fork. In the predominant state, the ring-shaped C-terminal ATPase of MCM is compact and contacts single-stranded DNA, via a set of pre-sensor 1 hairpins that spiral around the translocation substrate. In the second state, the ATPase module is relaxed and apparently substrate free, while DNA intimately contacts the downstream amino-terminal tier of the MCM motor ring. These results, supported by single-molecule FRET measurements, lead us to suggest a replication fork unwinding mechanism whereby the N-terminal and AAA+ tiers of the MCM work in concert to translocate on single-stranded DNA.
History
DepositionFeb 1, 2016-
Header (metadata) releaseFeb 10, 2016-
Map releaseFeb 24, 2016-
UpdateFeb 8, 2017-
Current statusFeb 8, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0895
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0895
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-3319.png

Downloads & links

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Map

FileDownload / File: emd_3319.map.gz / Format: CCP4 / Size: 5.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCMG treated with forked DNA substrate in the presence of ATPgS in a relaxed Mcm5-2 AAA+ configuration
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.7 Å/pix.
x 112 pix.
= 302.4 Å		2.7 Å/pix.
x 112 pix.
= 302.4 Å		2.7 Å/pix.
x 112 pix.
= 302.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.7 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0895 / Movie #1: 0.0895
Minimum - Maximum-0.05939161 - 0.22069867
Average (Standard dev.)0.00363789 (±0.01793187)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions112112112
Spacing112112112
CellA=B=C: 302.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.72.72.7
M x/y/z112112112
origin x/y/z0.0000.0000.000
length x/y/z302.400302.400302.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS112112112
D min/max/mean-0.0590.2210.004

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Supplemental data

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Sample components

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Entire : CMG treated with forked DNA substrate and ATPgS

EntireName: CMG treated with forked DNA substrate and ATPgS
Components
  • Sample: CMG treated with forked DNA substrate and ATPgS
  • Protein or peptide: Mcm2
  • Protein or peptide: Mcm3
  • Protein or peptide: Mcm4
  • Protein or peptide: Mcm5
  • Protein or peptide: Mcm6
  • Protein or peptide: Mcm7
  • Protein or peptide: Cdc45
  • Protein or peptide: Psf1
  • Protein or peptide: Psf2
  • Protein or peptide: Psf3
  • Protein or peptide: Sld5
  • DNA: Model replication DNA fork leading strand template
  • DNA: Model replication DNA fork lagging strand template

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Supramolecule #1000: CMG treated with forked DNA substrate and ATPgS

SupramoleculeName: CMG treated with forked DNA substrate and ATPgS / type: sample / ID: 1000 / Number unique components: 13
Molecular weightExperimental: 700 KDa / Theoretical: 700 KDa

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Macromolecule #1: Mcm2

MacromoleculeName: Mcm2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Drosophila melanogaster (fruit fly) / synonym: Fruit fly
Molecular weightExperimental: 100 KDa / Theoretical: 100 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper) / Recombinant cell: High Five / Recombinant plasmid: pFastBac-Mcm2
SequenceUniProtKB: DNA replication licensing factor Mcm2 / InterPro: DNA replication licensing factor Mcm2

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Macromolecule #4: Mcm3

MacromoleculeName: Mcm3 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Drosophila melanogaster (fruit fly) / synonym: Fruit fly
Molecular weightExperimental: 90 KDa / Theoretical: 90 KDa
Recombinant expressionOrganism: unidentified baculovirus / Recombinant plasmid: pFastBac-Mcm3
SequenceUniProtKB: DNA replication licensing factor Mcm3 / InterPro: DNA replication licensing factor Mcm3

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Macromolecule #5: Mcm4

MacromoleculeName: Mcm4 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Drosophila melanogaster (fruit fly) / synonym: Fruit fly
Molecular weightExperimental: 97 KDa / Theoretical: 97 KDa
Recombinant expressionOrganism: unidentified baculovirus / Recombinant plasmid: pFastBac-Mcm4
SequenceUniProtKB: DNA replication licensing factor MCM4 / InterPro: Mini-chromosome maintenance complex protein 4

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Macromolecule #6: Mcm5

MacromoleculeName: Mcm5 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Drosophila melanogaster (fruit fly) / synonym: Fruit fly
Molecular weightExperimental: 82 KDa / Theoretical: 82 KDa
Recombinant expressionOrganism: unidentified baculovirus / Recombinant plasmid: pFastBac-Mcm5
SequenceUniProtKB: DNA replication licensing factor Mcm5 / InterPro: DNA replication licensing factor Mcm5

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Macromolecule #7: Mcm6

MacromoleculeName: Mcm6 / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Drosophila melanogaster (fruit fly) / synonym: Fruit fly
Molecular weightExperimental: 92 KDa / Theoretical: 92 KDa
Recombinant expressionOrganism: unidentified baculovirus / Recombinant plasmid: pFastBac-Mcm6
SequenceUniProtKB: DNA replication licensing factor Mcm6 / InterPro: DNA replication licensing factor Mcm6

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Macromolecule #8: Mcm7

MacromoleculeName: Mcm7 / type: protein_or_peptide / ID: 8 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Drosophila melanogaster (fruit fly) / synonym: Fruit fly
Molecular weightExperimental: 81 KDa / Theoretical: 81 KDa
Recombinant expressionOrganism: unidentified baculovirus / Recombinant plasmid: pFastBac-Mcm7
SequenceUniProtKB: DNA replication licensing factor Mcm7 / InterPro: DNA replication licensing factor Mcm7

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Macromolecule #9: Cdc45

MacromoleculeName: Cdc45 / type: protein_or_peptide / ID: 9 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Drosophila melanogaster (fruit fly) / synonym: Fruit fly
Molecular weightExperimental: 66 KDa / Theoretical: 66 KDa
Recombinant expressionOrganism: unidentified baculovirus / Recombinant plasmid: pFastBac-Cdc45
SequenceUniProtKB: CDC45L / InterPro: CDC45 family

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Macromolecule #10: Psf1

MacromoleculeName: Psf1 / type: protein_or_peptide / ID: 10 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Drosophila melanogaster (fruit fly) / synonym: Fruit fly
Molecular weightExperimental: 23 KDa / Theoretical: 23 KDa
Recombinant expressionOrganism: unidentified baculovirus / Recombinant plasmid: pFastBac-Psf1
SequenceUniProtKB: DNA replication complex GINS protein PSF1 / InterPro: GINS subunit, domain A

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Macromolecule #11: Psf2

MacromoleculeName: Psf2 / type: protein_or_peptide / ID: 11 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Drosophila melanogaster (fruit fly) / synonym: Fruit fly
Molecular weightExperimental: 23 KDa / Theoretical: 23 KDa
Recombinant expressionOrganism: unidentified baculovirus / Recombinant plasmid: pFastBac-Psf2
SequenceUniProtKB: Probable DNA replication complex GINS protein PSF2
InterPro: DNA replication complex GINS protein Psf2

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Macromolecule #12: Psf3

MacromoleculeName: Psf3 / type: protein_or_peptide / ID: 12 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Drosophila melanogaster (fruit fly) / synonym: Fruit fly
Molecular weightExperimental: 25 KDa / Theoretical: 25 KDa
Recombinant expressionOrganism: unidentified baculovirus / Recombinant plasmid: pFastBac-Psf3
SequenceUniProtKB: DNA replication complex GINS protein PSF3 / InterPro: GINS complex, subunit Psf3

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Macromolecule #13: Sld5

MacromoleculeName: Sld5 / type: protein_or_peptide / ID: 13 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Drosophila melanogaster (fruit fly) / synonym: Fruit fly
Molecular weightExperimental: 26 KDa / Theoretical: 26 KDa
Recombinant expressionOrganism: unidentified baculovirus / Recombinant plasmid: pFastBac-Sld5
SequenceUniProtKB: DNA replication complex GINS protein SLD5 / InterPro: GINS complex subunit Sld5

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Macromolecule #2: Model replication DNA fork leading strand template

MacromoleculeName: Model replication DNA fork leading strand template / type: dna / ID: 2 / Classification: DNA / Structure: OTHER / Synthetic?: Yes
Source (natural)Organism: synthetic construct (others)
SequenceString:
CACTCGGGCT CGTTTTACAA CGTCGTGACT GGGCACTTGA TCGGCCAACC TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT

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Macromolecule #3: Model replication DNA fork lagging strand template

MacromoleculeName: Model replication DNA fork lagging strand template / type: dna / ID: 3 / Classification: DNA / Structure: OTHER / Synthetic?: Yes
Source (natural)Organism: synthetic construct (others)
SequenceString:
CTGGCGTCGG GTCGGCGGTT GGCCGATCAA GTGCCCAGTC ACGACGTTGT AAAACGAGCC CGAGTG

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.6
Details: 25 mM Hepes, 50 mM sodium acetate, 10 mM magnesium acetate, 1 mM DTT, 0.1mM ATPgammaS
GridDetails: Quantifoil 1.2/1.3 or C-flat 1/1
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV / Method: Blot for 4 seconds before plunging

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Electron microscopy #1

Microscopy ID1
MicroscopeFEI POLARA 300
Specialist opticsEnergy filter - Name: GIF / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
DateAug 7, 2015
Image recordingCategory: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Digitization - Sampling interval: 5 µm / Number real images: 2098 / Average electron dose: 48 e/Å2 / Details: data was recorded as movies of 25 frames / Bits/pixel: 32
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.7 µm / Nominal magnification: 37037
Sample stageSpecimen holder: Multi cartridge holder / Specimen holder model: OTHER
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Electron microscopy #2

Microscopy ID2
MicroscopeFEI POLARA 300
Specialist opticsEnergy filter - Name: GIF / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
DateAug 7, 2015
Image recordingCategory: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Digitization - Sampling interval: 5 µm / Number real images: 2098 / Average electron dose: 48 e/Å2 / Details: data was recorded as movies of 25 frames / Bits/pixel: 32
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.7 µm / Nominal magnification: 37037
Sample stageSpecimen holder: Multi cartridge holder / Specimen holder model: OTHER
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

DetailsParticles were picked in EMAN2; Contrast Transfer Function was estimated using CTFFIND4. All further processing was performed within the RELION 1.4 environment.
CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 9.8 Å / Resolution method: OTHER / Software - Name: RELION1.4 / Number images used: 13692
FSC plot (resolution estimation)

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