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- EMDB-3318: CryoEM structure of the CMG replicative helicase bound to a DNA f... -

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Entry
Database: EMDB / ID: 3318
TitleCryoEM structure of the CMG replicative helicase bound to a DNA fork (compact state)
Map dataCMG treated with forked DNA substrate in the presence of ATPgS in a compact Mcm5-2 AAA+ configuration
SampleCMG bound to DNA fork and ATPgS treated:
Mcm2 / (nucleic-acidNucleic acid) x 2 / Mcm3 / Mcm4 / Mcm5 / Mcm6 / Mcm7 / Cdc45 / Psf1 / Psf2 ...Mcm2 / (nucleic-acidNucleic acid) x 2 / Mcm3 / Mcm4 / Mcm5 / Mcm6 / Mcm7 / Cdc45 / Psf1 / Psf2 / Psf3 / Sld5
KeywordsCdc45 / GINS / MCM / CMG / helicase / DNA replication
Function / homologyGINS, helical bundle-like domain superfamily / GINS complex protein / GINS complex, subunit Psf3 / GINS complex subunit Sld5 / DNA replication licensing factor Mcm7 / DNA replication licensing factor Mcm6 / DNA replication licensing factor Mcm5 / Mini-chromosome maintenance complex protein 4 / DNA replication licensing factor Mcm3 / CDC45-like protein ...GINS, helical bundle-like domain superfamily / GINS complex protein / GINS complex, subunit Psf3 / GINS complex subunit Sld5 / DNA replication licensing factor Mcm7 / DNA replication licensing factor Mcm6 / DNA replication licensing factor Mcm5 / Mini-chromosome maintenance complex protein 4 / DNA replication licensing factor Mcm3 / CDC45-like protein / MCM N-terminal domain / MCM P-loop domain / GINS complex, subunit Psf3 superfamily / DNA replication licensing factor Mcm2 / DNA replication complex GINS protein Psf2 / GINS complex, subunit Psf1 / CDC45 family / AAA+ ATPase domain / MCM domain / Mini-chromosome maintenance protein 2 / Nucleic acid-binding, OB-fold / GINS complex protein Sld5, alpha-helical domain / Unwinding of DNA / MCM OB domain / DNA replication complex GINS protein SLD5, C-terminal / Switching of origins to a post-replicative state / Mini-chromosome maintenance protein / Activation of the pre-replicative complex / MCM N-terminal domain / Orc1 removal from chromatin / Mini-chromosome maintenance, conserved site / Assembly of the pre-replicative complex / Activation of ATR in response to replication stress / P-loop containing nucleoside triphosphate hydrolase / MCM family domain profile. / MCM family signature. / MCM OB domain / DNA replication complex GINS protein SLD5 C-terminus / GINS subunit, domain A / DNA endoreduplication / DNA amplification / eggshell chorion gene amplification / negative regulation of DNA helicase activity / CDC45 family / DNA strand elongation involved in mitotic DNA replication / DNA replication complex GINS protein Psf2 / GINS complex, subunit Psf3 / GINS complex / GINS subunit, domain A / GINS complex subunit Sld5 / mitotic DNA replication preinitiation complex assembly / DNA replication licensing factor Mcm3 / DNA replication licensing factor Mcm7 / DNA replication licensing factor Mcm5 / regulation of chromatin silencing at telomere / DNA replication licensing factor Mcm6 / Mini-chromosome maintenance complex protein 4 / DNA replication licensing factor Mcm2 / MCM complex / nuclear pre-replicative complex / DNA replication preinitiation complex / 3'-5' DNA helicase activity / double-strand break repair via break-induced replication / regulation of DNA-templated transcription, elongation / resolution of meiotic recombination intermediates / replication fork protection complex / DNA strand elongation involved in DNA replication / chromosome condensation / DNA duplex unwinding / DNA helicase activity / DNA replication origin binding / DNA replication initiation / positive regulation of G1/S transition of mitotic cell cycle / DNA helicase / mitotic spindle organization / meiotic cell cycle / helicase activity / single-stranded DNA binding / DNA-dependent DNA replication / mitotic cell cycle / DNA replication / cell cycle / cell division / chromatin binding / DNA binding / ATP binding / nucleus / metal ion binding / cytosol / cytoplasm / Probable DNA replication complex GINS protein PSF2 / DNA replication licensing factor Mcm7 / AT18545p / IP07275p / CDC45L / DNA replication licensing factor Mcm5 / DNA replication complex GINS protein SLD5 / DNA replication licensing factor Mcm6 / DNA replication licensing factor MCM4 / DNA replication licensing factor Mcm2
Function and homology information
SourceDrosophila melanogaster (fruit fly) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / 7.4 Å resolution
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
DateDeposition: Feb 1, 2016 / Header (metadata) release: Feb 10, 2016 / Map release: Feb 24, 2016 / Last update: Feb 8, 2017

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.11
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.11
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_3318.map.gz (map file in CCP4 format, 5489 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
112 pix
2.7 Å/pix.
= 302.4 Å
112 pix
2.7 Å/pix.
= 302.4 Å
112 pix
2.7 Å/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
Contour Level:0.11 (by author), 0.11 (movie #1):
Minimum - Maximum-0.62580413 - 0.69123393
Average (Standard dev.)0.00087016 (0.02675519)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions112112112
Origin000
Limit111111111
Spacing112112112
CellA=B=C: 302.4 Å
α=β=γ: 90.0 deg.

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
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS112112112
D min/max/mean-0.6260.6910.001

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Supplemental data

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Sample components

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Entire CMG bound to DNA fork and ATPgS treated

EntireName: CMG bound to DNA fork and ATPgS treated / Number of components: 13
MassTheoretical: 700 kDa / Experimental: 700 kDa

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Component #1: protein, Mcm2

ProteinName: Mcm2 / Oligomeric Details: Monomer / Recombinant expression: Yes / Number of Copies: 1
MassTheoretical: 100 kDa / Experimental: 100 kDa
SourceSpecies: Drosophila melanogaster (fruit fly)
Source (engineered)Expression System: Trichoplusia (insect) / Vector: pFastBac-Mcm2 / Cell of expression system: High Five
External referencesUniProt: DNA replication licensing factor Mcm2 / InterPro: DNA replication licensing factor Mcm2

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Component #2: nucleic-acid, Model replication DNA fork leading strand template

Nucleic-acidName: Model replication DNA fork leading strand template / Class: DNA / Structure: OTHER / Synthetic: Yes
Sequence:
CACTCGGGCT CGTTTTACAA CGTCGTGACT GGGCACTTGA TCGGCCAACC TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT
SourceSpecies: synthetic construct (others)

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Component #3: nucleic-acid, Model replication DNA fork lagging strand template

Nucleic-acidName: Model replication DNA fork lagging strand template / Class: DNA / Structure: OTHER / Synthetic: Yes
Sequence:
CTGGCGTCGG GTCGGCGGTT GGCCGATCAA GTGCCCAGTC ACGACGTTGT AAAACGAGCC CGAGTG
SourceSpecies: synthetic construct (others)

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Component #4: protein, Mcm3

ProteinName: Mcm3 / Oligomeric Details: Monomer / Recombinant expression: Yes / Number of Copies: 1
MassTheoretical: 90 kDa / Experimental: 90 kDa
SourceSpecies: Drosophila melanogaster (fruit fly)
Source (engineered)Expression System: unidentified baculovirus / Vector: pFastBac-Mcm3
External referencesInterPro: DNA replication licensing factor Mcm3 / UniProt: DNA replication licensing factor Mcm3

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Component #5: protein, Mcm4

ProteinName: Mcm4 / Oligomeric Details: Monomer / Recombinant expression: Yes / Number of Copies: 1
MassTheoretical: 97 kDa / Experimental: 97 kDa
SourceSpecies: Drosophila melanogaster (fruit fly)
Source (engineered)Expression System: unidentified baculovirus / Vector: pFastBac-Mcm4
External referencesInterPro: Mini-chromosome maintenance complex protein 4 / UniProt: DNA replication licensing factor MCM4

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Component #6: protein, Mcm5

ProteinName: Mcm5 / Oligomeric Details: Monomer / Recombinant expression: Yes / Number of Copies: 1
MassTheoretical: 82 kDa / Experimental: 82 kDa
SourceSpecies: Drosophila melanogaster (fruit fly)
Source (engineered)Expression System: unidentified baculovirus / Vector: pFastBac-Mcm5
External referencesInterPro: DNA replication licensing factor Mcm5 / UniProt: DNA replication licensing factor Mcm5

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Component #7: protein, Mcm6

ProteinName: Mcm6 / Oligomeric Details: Monomer / Number of Copies: 1 / Recombinant expression: Yes
MassTheoretical: 92 kDa / Experimental: 92 kDa
SourceSpecies: Drosophila melanogaster (fruit fly)
Source (engineered)Expression System: unidentified baculovirus / Vector: pFastBac-Mcm6
External referencesUniProt: DNA replication licensing factor Mcm6 / InterPro: DNA replication licensing factor Mcm6

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Component #8: protein, Mcm7

ProteinName: Mcm7 / Oligomeric Details: Monomer / Recombinant expression: Yes / Number of Copies: 1
MassTheoretical: 81 kDa / Experimental: 81 kDa
SourceSpecies: Drosophila melanogaster (fruit fly)
Source (engineered)Expression System: unidentified baculovirus / Vector: pFastBac-Mcm7
External referencesUniProt: DNA replication licensing factor Mcm7 / InterPro: DNA replication licensing factor Mcm7

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Component #9: protein, Cdc45

ProteinName: Cdc45 / Oligomeric Details: Monomer / Number of Copies: 1 / Recombinant expression: Yes
MassTheoretical: 66 kDa / Experimental: 66 kDa
SourceSpecies: Drosophila melanogaster (fruit fly)
Source (engineered)Expression System: unidentified baculovirus / Vector: pFastBac-Cdc45
External referencesInterPro: CDC45 family / UniProt: CDC45L

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Component #10: protein, Psf1

ProteinName: Psf1 / Oligomeric Details: Monomer / Number of Copies: 1 / Recombinant expression: Yes
MassTheoretical: 23 kDa / Experimental: 23 kDa
SourceSpecies: Drosophila melanogaster (fruit fly)
Source (engineered)Expression System: unidentified baculovirus / Vector: pFastBac-Psf1
External referencesUniProt: IP07275p / InterPro: GINS subunit, domain A

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Component #11: protein, Psf2

ProteinName: Psf2 / Oligomeric Details: Monomer / Number of Copies: 1 / Recombinant expression: Yes
MassTheoretical: 23 kDa / Experimental: 23 kDa
SourceSpecies: Drosophila melanogaster (fruit fly)
Source (engineered)Expression System: unidentified baculovirus / Vector: pFastBac-Psf2
External referencesUniProt: Probable DNA replication complex GINS protein PSF2 / InterPro: DNA replication complex GINS protein Psf2

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Component #12: protein, Psf3

ProteinName: Psf3 / Oligomeric Details: Monomer / Recombinant expression: Yes / Number of Copies: 1
MassTheoretical: 25 kDa / Experimental: 25 kDa
SourceSpecies: Drosophila melanogaster (fruit fly)
Source (engineered)Expression System: unidentified baculovirus / Vector: pFastBac-Psf3
External referencesUniProt: AT18545p / InterPro: GINS complex, subunit Psf3

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Component #13: protein, Sld5

ProteinName: Sld5 / Oligomeric Details: Monomer / Number of Copies: 1 / Recombinant expression: Yes
MassTheoretical: 26 kDa / Experimental: 26 kDa
SourceSpecies: Drosophila melanogaster (fruit fly)
Source (engineered)Expression System: unidentified baculovirus / Vector: pFastBac-Sld5
External referencesInterPro: GINS complex subunit Sld5 / UniProt: DNA replication complex GINS protein SLD5

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionBuffer solution: 25 mM Hepes, 50 mM sodium acetate, 10 mM magnesium acetate, 1 mM DTT, 0.1mM ATPgammaS
pH: 7.6
Support filmQuantifoil 1.2/1.3 or C-flat 1/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Method: Blot for 4 seconds before plunging

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Electron microscopy imaging #1

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
ImagingMicroscope: FEI POLARA 300 / Date: Aug 7, 2015
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 48 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 37037 X (nominal) / Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 1700 - 3500 nm / Energy filter: GIF / Energy window: 0-20 eV
Specimen HolderHolder: Multi cartridge holder / Model: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Electron microscopy imaging #2

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
ImagingMicroscope: FEI POLARA 300 / Date: Aug 7, 2015
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 48 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 37037 X (nominal) / Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 1700 - 3500 nm / Energy filter: GIF / Energy window: 0-20 eV
Specimen HolderHolder: Multi cartridge holder / Model: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 2098 / Sampling size: 5 microns / Bit depth: 32 / Details: data was recorded as movies of 25 frames

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 38792
Details: Particles were picked in EMAN2; Contrast Transfer Function was estimated using CTFFIND4. All further processing was performed within the RELION 1.4 environment.
3D reconstructionSoftware: RELION1.4 / CTF correction: Each particle / Resolution: 7.4 Å / Resolution method: FSC 0.143, gold-standard
FSC plot
(resolution estimation)

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