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- EMDB-2772: CMG helicase bound to DNA and ATPgS -

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Basic information

Entry
Database: EMDB / ID: EMD-2772
TitleCMG helicase bound to DNA and ATPgS
Map dataReconstruction of the recombinant Drosophila CMG helicase bound to ATPgS and DNA
Sample
  • Sample: Drosophila melanogaster Cdc45-Mcm2-7-GINS complex bound to ATPgS and DNA
  • Protein or peptide: x 11 types
  • DNA: x 2 types
KeywordsHelicase / AAA+ ATPase / DNA replication
Function / homology
Function and homology information


Unwinding of DNA / Switching of origins to a post-replicative state / DNA endoreduplication / Assembly of the pre-replicative complex / 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 / Switching of origins to a post-replicative state / DNA endoreduplication / Assembly of the pre-replicative complex / 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 / MCM complex / DNA replication preinitiation 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 / meiotic cell cycle / regulation of DNA-templated transcription elongation / helicase activity / single-stranded DNA binding / mitotic cell cycle / DNA helicase / DNA replication / cell division / chromatin binding / ATP hydrolysis activity / ATP binding / metal ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
CDC45 family / CDC45 family / CDC45-like protein / DNA replication complex GINS protein Psf2 / DNA replication complex GINS protein Psf2 / 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 ...CDC45 family / CDC45 family / CDC45-like protein / DNA replication complex GINS protein Psf2 / DNA replication complex GINS protein Psf2 / 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 / MCM4, winged helix 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 / MCM OB domain / Mini-chromosome maintenance protein / MCM, AAA-lid domain / MCM P-loop 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) / unidentified (others)
Methodsingle particle reconstruction / negative staining / Resolution: 17.8 Å
AuthorsCosta A / Renault L / Swuec P / Petojevic T / Pesavento JJ / Ilves I / MacLellan-Gibson K / Fleck RA / Botchan MR / Berger JM
CitationJournal: Elife / Year: 2014
Title: DNA binding polarity, dimerization, and ATPase ring remodeling in the CMG helicase of the eukaryotic replisome.
Authors: Alessandro Costa / Ludovic Renault / Paolo Swuec / Tatjana Petojevic / James J Pesavento / Ivar Ilves / Kirsty MacLellan-Gibson / Roland A Fleck / Michael R Botchan / James M Berger /
Abstract: The Cdc45/Mcm2-7/GINS (CMG) helicase separates DNA strands during replication in eukaryotes. How the CMG is assembled and engages DNA substrates remains unclear. Using electron microscopy, we have ...The Cdc45/Mcm2-7/GINS (CMG) helicase separates DNA strands during replication in eukaryotes. How the CMG is assembled and engages DNA substrates remains unclear. Using electron microscopy, we have determined the structure of the CMG in the presence of ATPγS and a DNA duplex bearing a 3' single-stranded tail. The structure shows that the MCM subunits of the CMG bind preferentially to single-stranded DNA, establishes the polarity by which DNA enters into the Mcm2-7 pore, and explains how Cdc45 helps prevent DNA from dissociating from the helicase. The Mcm2-7 subcomplex forms a cracked-ring, right-handed spiral when DNA and nucleotide are bound, revealing unexpected congruencies between the CMG and both bacterial DnaB helicases and the AAA+ motor of the eukaryotic proteasome. The existence of a subpopulation of dimeric CMGs establishes the subunit register of Mcm2-7 double hexamers and together with the spiral form highlights how Mcm2-7 transitions through different conformational and assembly states as it matures into a functional helicase.
History
DepositionAug 28, 2014-
Header (metadata) releaseSep 10, 2014-
Map releaseSep 10, 2014-
UpdateSep 10, 2014-
Current statusSep 10, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.37
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.37
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2772-dep...

Downloads & links

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Map

FileDownload / File: emd_2772.map.gz / Format: CCP4 / Size: 17.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the recombinant Drosophila CMG helicase bound to ATPgS and DNA
Voxel sizeX=Y=Z: 2.14 Å
Density
Contour LevelBy AUTHOR: 0.37 / Movie #1: 0.37
Minimum - Maximum-0.32566851 - 0.81758922
Average (Standard dev.)0.00992203 (±0.0676895)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions168168168
Spacing168168168
CellA=B=C: 359.52002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.142.142.14
M x/y/z168168168
origin x/y/z0.0000.0000.000
length x/y/z359.520359.520359.520
α/β/γ90.00090.00090.000
start NX/NY/NZ-40-32-96
NX/NY/NZ8165193
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS168168168
D min/max/mean-0.3260.8180.010

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Supplemental data

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Sample components

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Entire : Drosophila melanogaster Cdc45-Mcm2-7-GINS complex bound to ATPgS ...

EntireName: Drosophila melanogaster Cdc45-Mcm2-7-GINS complex bound to ATPgS and DNA
Components
  • Sample: Drosophila melanogaster Cdc45-Mcm2-7-GINS complex bound to ATPgS and DNA
  • 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: LEAD60
  • DNA: 3BTNLAG20

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Supramolecule #1000: Drosophila melanogaster Cdc45-Mcm2-7-GINS complex bound to ATPgS ...

SupramoleculeName: Drosophila melanogaster Cdc45-Mcm2-7-GINS complex bound to ATPgS and DNA
type: sample / ID: 1000 / Details: The sample was monodisperse / Oligomeric state: 13-mer / Number unique components: 13
Molecular weightTheoretical: 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 / Organelle: Nucleus
Molecular weightTheoretical: 100 KDa
Recombinant expressionOrganism: unidentified baculovirus / Recombinant plasmid: pFastBac-Mcm2
SequenceUniProtKB: DNA replication licensing factor Mcm2 / InterPro: DNA replication licensing factor Mcm2

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Macromolecule #2: Mcm3

MacromoleculeName: Mcm3 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Drosophila melanogaster (fruit fly) / synonym: Fruit fly / Organelle: Nucleus
Molecular weightTheoretical: 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 #3: Mcm4

MacromoleculeName: Mcm4 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Drosophila melanogaster (fruit fly) / synonym: Fruit fly / Organelle: Nucleus
Molecular weightTheoretical: 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 #4: Mcm5

MacromoleculeName: Mcm5 / 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 / Organelle: Nucleus
Molecular weightTheoretical: 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 #5: Mcm6

MacromoleculeName: Mcm6 / 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 / Organelle: Nucleus
Molecular weightTheoretical: 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 #6: Mcm7

MacromoleculeName: Mcm7 / 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 / Organelle: Nucleus
Molecular weightTheoretical: 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 #7: Cdc45

MacromoleculeName: Cdc45 / type: protein_or_peptide / ID: 7 / Name.synonym: Cdc45L / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Drosophila melanogaster (fruit fly) / synonym: Fruit fly / Organelle: Nucleus
Molecular weightTheoretical: 66 KDa
Recombinant expressionOrganism: unidentified baculovirus / Recombinant plasmid: pFastBac-Cdc45
SequenceUniProtKB: CDC45L / InterPro: CDC45 family

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Macromolecule #8: Psf1

MacromoleculeName: Psf1 / 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 / Organelle: Nucleus
Molecular weightTheoretical: 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 #9: Psf2

MacromoleculeName: Psf2 / 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 / Organelle: Nucleus
Molecular weightTheoretical: 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 #10: Psf3

MacromoleculeName: Psf3 / 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 / Organelle: Nucleus
Molecular weightTheoretical: 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 #11: Sld5

MacromoleculeName: Sld5 / 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 / Organelle: Nucleus
Molecular weightTheoretical: 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 #12: LEAD60

MacromoleculeName: LEAD60 / type: dna / ID: 12
Details: The two oligonucleotides have been annealed to obtain a 20mer duplex DNA with a 40mer 3' overhang.
Classification: DNA / Structure: OTHER / Synthetic?: Yes
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 18.271 KDa
SequenceString:
GGGCACTTGA TCGGCCAACC TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT

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Macromolecule #13: 3BTNLAG20

MacromoleculeName: 3BTNLAG20 / type: dna / ID: 13
Details: The two oligonucleotides have been annealed to obtain a 20mer duplex DNA with a 40mer 3' overhang. 3BTNLAG20 contains a biotin on the 3' end (duplex end).
Classification: DNA / Structure: OTHER / Synthetic?: Yes
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 6.569 KDa
SequenceString:
GGTTGGCCGA TCAAGTGCCC

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.02 mg/mL
BufferpH: 7.6
Details: 25 mM HEPES , 50 mM sodium acetate, 10 mM magnesium acetate, 0.1 mM ATPgS, 1 mM DTT
StainingType: NEGATIVE
Details: The grid with adsorbed protein was floated for 10 seconds on four consecutive 75 microliter drops of 2% w/v uranyl formate solution.
GridDetails: 400 mesh copper grids with thin carbon support.
VitrificationCryogen name: NONE / Instrument: OTHER
Details: The sample stained with a fresh 2% (wt/vol) uranyl formate solution.

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Electron microscopy

MicroscopeJEOL 2100
Electron beamAcceleration voltage: 200 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 70000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 50000
Sample stageSpecimen holder: single tilt room temperature / Specimen holder model: JEOL
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 60,000 times magnification
DateMay 8, 2013
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 579 / Average electron dose: 35 e/Å2 / Bits/pixel: 32

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Image processing

CTF correctionDetails: EMAN2
Final two d classificationNumber classes: 100
Final angle assignmentDetails: SPARX
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 17.8 Å / Resolution method: OTHER / Software - Name: EMAN2, SPARX / Number images used: 29913
DetailsParticles were selected semi-automatically using e2boxer

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