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- PDB-6raw: D. melanogaster CMG-DNA, State 1A -

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Entry
Database: PDB / ID: 6raw
TitleD. melanogaster CMG-DNA, State 1A
Components
  • (DNA replication licensing factor ...) x 6
  • AT18545p
  • CDC45LCDC45-related protein
  • DNA (5'-D(P*TP*CP*GP*AP*TP*CP*GP*AP*TP*CP*GP*AP*T)-3')
  • DNA replication complex GINS protein SLD5
  • DNA
  • IP07275p
  • Probable DNA replication complex GINS protein PSF2
KeywordsREPLICATION / Helicase / ATPase / AAA+ / DNA unwinding
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
Ribosomal Protein L9; domain 1 - #60 / Methane Monooxygenase Hydroxylase; Chain G, domain 1 - #1030 / Ribosomal Protein L9; domain 1 - #50 / Ribosomal Protein L9; domain 1 / CDC45 family / CDC45-like protein / DNA replication complex GINS protein Psf2 / GINS complex, subunit Psf1 / GINS complex, subunit Psf3 / GINS complex, subunit Psf3 superfamily ...Ribosomal Protein L9; domain 1 - #60 / Methane Monooxygenase Hydroxylase; Chain G, domain 1 - #1030 / Ribosomal Protein L9; domain 1 - #50 / Ribosomal Protein L9; domain 1 / CDC45 family / CDC45-like protein / DNA replication complex GINS protein Psf2 / 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 protein / MCM4, winged helix domain / 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 / Mcm6, C-terminal winged-helix domain / MCM6 C-terminal winged-helix domain / 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 / Methane Monooxygenase Hydroxylase; Chain G, domain 1 / Winged helix-like DNA-binding domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Nucleic acid-binding, OB-fold / Up-down Bundle / P-loop containing nucleoside triphosphate hydrolase / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / 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 ...ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsEickhoff, P. / Martino, F. / 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 single- ...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.
History
DepositionApr 8, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 11, 2019Provider: repository / Type: Initial release
Revision 1.1Sep 18, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

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Assembly

Deposited unit
2: DNA replication licensing factor Mcm2
3: DNA replication licensing factor Mcm3
4: DNA replication licensing factor MCM4
5: DNA replication licensing factor Mcm5
6: DNA replication licensing factor Mcm6
7: DNA replication licensing factor Mcm7
F: DNA
G: DNA (5'-D(P*TP*CP*GP*AP*TP*CP*GP*AP*TP*CP*GP*AP*T)-3')
A: CDC45L
H: IP07275p
L: Probable DNA replication complex GINS protein PSF2
M: AT18545p
N: DNA replication complex GINS protein SLD5
hetero molecules


Theoretical massNumber of molelcules
Total (without water)722,68319
Polymers719,87913
Non-polymers2,8036
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area64680 Å2
ΔGint-293 kcal/mol
Surface area252700 Å2
MethodPISA

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Components

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DNA replication licensing factor ... , 6 types, 6 molecules 234567

#1: Protein DNA replication licensing factor Mcm2 / Minichromosome maintenance 2 protein / DmMCM2


Mass: 100537.438 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly) / Gene: Mcm2, CG7538 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P49735, DNA helicase
#2: Protein DNA replication licensing factor Mcm3 / Minichromosome maintenance 3 protein / DmMCM3


Mass: 91045.164 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly) / Gene: Mcm3, Mcm3-RA, CG4206 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9XYU1, DNA helicase
#3: Protein DNA replication licensing factor MCM4 / Protein disc proliferation abnormal


Mass: 96735.094 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly) / Gene: dpa, CG1616 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q26454, DNA helicase
#4: Protein DNA replication licensing factor Mcm5 / Minichromosome maintenance 5 protein / DmMCM5


Mass: 82375.320 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly) / Gene: Mcm5, CG4082 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9VGW6, DNA helicase
#5: Protein DNA replication licensing factor Mcm6 / DmMCM6


Mass: 92467.820 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly) / Gene: Mcm6, CG4039 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9V461, DNA helicase
#6: Protein DNA replication licensing factor Mcm7 / Minichromosome maintenance 7 protein / DmMCM3


Mass: 81399.352 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly) / Gene: Mcm7, CG4978 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9XYU0, DNA helicase

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DNA chain , 2 types, 2 molecules FG

#7: DNA chain DNA /


Mass: 7930.117 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Drosophila melanogaster (fruit fly)
#8: DNA chain DNA (5'-D(P*TP*CP*GP*AP*TP*CP*GP*AP*TP*CP*GP*AP*T)-3')


Mass: 3966.597 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Drosophila melanogaster (fruit fly)

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Protein , 5 types, 5 molecules AHLMN

#9: Protein CDC45L / CDC45-related protein / EG:BACR7A4.11 protein / LD35753p


Mass: 65968.789 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly)
Gene: CDC45L, anon-1Ec, CDC45, Cdc45, cdc45, D, dCDC45, dCDC45L, DmCdc45, Dmel\CG3658, EG:BACR7A4.11, CG3658, Dmel_CG3658
Production host: Trichoplusia ni (cabbage looper) / References: UniProt: O96989
#10: Protein IP07275p / Psf1


Mass: 23333.693 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly)
Gene: Psf1, CG9187-PA, Dmel\CG9187, psf1, CG9187, Dmel_CG9187
Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9W0I7
#11: Protein Probable DNA replication complex GINS protein PSF2 / GINS complex subunit 2


Mass: 23141.781 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly) / Gene: Psf2, CG18013 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9VQY9
#12: Protein AT18545p / CG2222-PA / Psf3 / isoform A / isoform B / isoform C


Mass: 24829.984 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly) / Gene: Psf3, Dmel\CG2222, CG2222, Dmel_CG2222 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9W2V7
#13: Protein DNA replication complex GINS protein SLD5


Mass: 26148.234 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly)
Gene: Sld5, anon-WO0172774.61, Dmel\CG14549, CG14549, Dmel_CG14549
Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9VBI1

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Non-polymers , 2 types, 6 molecules

#14: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#15: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1CMG helicase bound to forked DNA in the presence of ATP, State 1ACOMPLEX#1-#130MULTIPLE SOURCES
2CMG helicaseCOMPLEX#1-#6, #9-#131RECOMBINANT
3DNACOMPLEX#7-#81RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Drosophila melanogaster (fruit fly)7227
33Drosophila melanogaster (fruit fly)7227
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Trichoplusia ni (cabbage looper)7111
33synthetic construct (others)32630
Buffer solutionpH: 7.6
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 170329 / Symmetry type: POINT

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