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- PDB-8rig: Cryo-EM structure of an MCM helicase single hexamer loaded onto dsDNA. -

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Basic information

Entry
Database: PDB / ID: 8rig
TitleCryo-EM structure of an MCM helicase single hexamer loaded onto dsDNA.
Components
  • (DNA replication licensing factor ...) x 5
  • DNA (5'-D(P*AP*TP*GP*CP*AP*TP*GP*CP*AP*TP*GP*CP*AP*TP*GP*CP*AP*T)-3')
  • DNA (5'-D(P*GP*CP*AP*TP*GP*CP*AP*TP*GP*CP*AP*TP*GP*CP*AP*TP*GP*CP*AP*T)-3')
  • Minichromosome maintenance protein 5
KeywordsREPLICATION / MCM helicase / DNA replication
Function / homology
Function and homology information


MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / MCM complex binding / nuclear DNA replication / premeiotic DNA replication / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / mitotic DNA replication / Activation of the pre-replicative complex / CMG complex ...MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / MCM complex binding / nuclear DNA replication / premeiotic DNA replication / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / mitotic DNA replication / Activation of the pre-replicative complex / CMG complex / nuclear pre-replicative complex / Activation of ATR in response to replication stress / DNA replication preinitiation complex / MCM complex / replication fork protection complex / double-strand break repair via break-induced replication / single-stranded DNA helicase activity / mitotic DNA replication initiation / regulation of DNA-templated DNA replication initiation / silent mating-type cassette heterochromatin formation / DNA strand elongation involved in DNA replication / nuclear replication fork / DNA replication origin binding / DNA replication initiation / subtelomeric heterochromatin formation / helicase activity / transcription elongation by RNA polymerase II / heterochromatin formation / single-stranded DNA binding / DNA helicase / forked DNA-dependent helicase activity / single-stranded 3'-5' DNA helicase activity / four-way junction helicase activity / double-stranded DNA helicase activity / chromosome, telomeric region / DNA replication / DNA damage response / chromatin binding / ATP hydrolysis activity / zinc ion binding / nucleoplasm / ATP binding / nucleus / cytoplasm
Similarity search - Function
: / MCM3 winged helix domain / MCM4, winged helix domain / : / MCM5, C-terminal domain / DNA replication licensing factor Mcm5 / MCM3-like, winged helix domain / DNA replication licensing factor Mcm3 / Mini-chromosome maintenance complex protein 4 / DNA replication licensing factor Mcm6 ...: / MCM3 winged helix domain / MCM4, winged helix domain / : / MCM5, C-terminal domain / DNA replication licensing factor Mcm5 / MCM3-like, winged helix domain / 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 / Winged helix-like DNA-binding domain superfamily / Nucleic acid-binding, OB-fold / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / DNA replication licensing factor MCM3 / DNA replication licensing factor MCM2 / Minichromosome maintenance protein 5 / DNA replication licensing factor MCM4 / DNA replication licensing factor MCM7 / DNA replication licensing factor MCM6
Similarity search - Component
Biological speciesSaccharomyces cerevisiae S288C (yeast)
Saccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.41 Å
AuthorsMiller, T.C.R. / Lim, C.T. / Diffley, J.F.X. / Costa, A.
Funding support United Kingdom, European Union, Denmark, 6items
OrganizationGrant numberCountry
Wellcome TrustFC001065 United Kingdom
Medical Research Council (MRC, United Kingdom)FC001065 United Kingdom
Cancer Research UKFC001065 United Kingdom
European Research Council (ERC)820102European Union
Novo Nordisk FoundationNNF22OC0073571 Denmark
Danish National Research FoundationDNRF115 Denmark
CitationJournal: To Be Published
Title: Inhibition of Origin Recognition Complex by Cyclin Dependent Kinase Reveals Alternate Mechanism of MCM Loading
Authors: Miller, T.C.R. / Lim, C.T. / Diffley, J.F.X. / Costa, A.
History
DepositionDec 18, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 1, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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: Minichromosome maintenance protein 5
6: DNA replication licensing factor MCM6
7: DNA replication licensing factor MCM7
X: DNA (5'-D(P*GP*CP*AP*TP*GP*CP*AP*TP*GP*CP*AP*TP*GP*CP*AP*TP*GP*CP*AP*T)-3')
Y: DNA (5'-D(P*AP*TP*GP*CP*AP*TP*GP*CP*AP*TP*GP*CP*AP*TP*GP*CP*AP*T)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)624,64825
Polymers621,4528
Non-polymers3,19617
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area56660 Å2
ΔGint-317 kcal/mol
Surface area169000 Å2
MethodPISA

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Components

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DNA replication licensing factor ... , 5 types, 5 molecules 23467

#1: Protein DNA replication licensing factor MCM2 / Minichromosome maintenance protein 2


Mass: 98911.539 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: MCM2, YBL023C, YBL0438 / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P29469, DNA helicase
#2: Protein DNA replication licensing factor MCM3 / Minichromosome maintenance protein 3


Mass: 111720.242 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: MCM3 contains a CBP-TEV tag at the N-terminus: KRRWKKNFIAVSAANRFKKISSSGALENLYFQG
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: MCM3, YEL032W, SYGP-ORF23 / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P24279, DNA helicase
#3: Protein DNA replication licensing factor MCM4 / Cell division control protein 54


Mass: 105138.375 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: MCM4, CDC54, HCD21, YPR019W, YP9531.13 / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P30665, DNA helicase
#5: Protein DNA replication licensing factor MCM6 / Minichromosome maintenance protein 6


Mass: 113110.211 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: MCM6, YGL201C / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P53091, DNA helicase
#6: Protein DNA replication licensing factor MCM7 / Cell division control protein 47 / Minichromosome maintenance protein 7


Mass: 95049.875 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: MCM7, CDC47, YBR202W, YBR1441 / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P38132, DNA helicase

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Protein , 1 types, 1 molecules 5

#4: Protein Minichromosome maintenance protein 5 / Cell division control protein 46


Mass: 86505.734 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: MCM5, CDC46, YLR274W, L9328.1 / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P29496, DNA helicase

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DNA chain , 2 types, 2 molecules XY

#7: DNA chain DNA (5'-D(P*GP*CP*AP*TP*GP*CP*AP*TP*GP*CP*AP*TP*GP*CP*AP*TP*GP*CP*AP*T)-3')


Mass: 5804.773 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae (brewer's yeast)
#8: DNA chain DNA (5'-D(P*AP*TP*GP*CP*AP*TP*GP*CP*AP*TP*GP*CP*AP*TP*GP*CP*AP*T)-3')


Mass: 5211.398 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae (brewer's yeast)

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Non-polymers , 4 types, 17 molecules

#9: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#10: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#11: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#12: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 4 / 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
Has protein modificationN

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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

ComponentName: Hexameric MCM helicase complex loaded onto duplex DNA / Type: COMPLEX
Details: Structural intermediate on path to MCM-double hexamer formation during the process of origin licensing.
Entity ID: #1-#8 / Source: RECOMBINANT
Molecular weightValue: 0.61 MDa
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.6
SpecimenConc.: 0.05 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Complete biochemical reaction containing the MCM helicase in complex with Cdt1, the helicase loader (the origin recognition complex), the co-factor Cdc6, an origin or replication (DNA) ...Details: Complete biochemical reaction containing the MCM helicase in complex with Cdt1, the helicase loader (the origin recognition complex), the co-factor Cdc6, an origin or replication (DNA) flanked by nucleosomes, the regulatory kinase CDK and an inhibitor of CDK (Sic1). The sample was incubated with ATP yielding a range of structural intermediates in the MCM loading pathway.
Specimen supportDetails: Glow discharge at 40 mA for 5 minutes prior to applying graphene oxide.
Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 K

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 4500 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Image recordingElectron dose: 51.2 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 24506
EM imaging opticsEnergyfilter slit width: 20 eV
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

EM software
IDNameVersionCategoryDetails
1Topazparticle selectionTopaz picking performed as in Relion3.1. Iterative rounds of Topaz picking, particle selection and training to optimise model.
2EPUimage acquisition
7Coot0.9.5model fitting
11RELION3.1classification
12RELION3.13D reconstruction
13PHENIX1.19.2_4158:model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 621909
3D reconstructionResolution: 3.41 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 396366
Details: Reported resolution obtained from maps after Relion 3D Refinement
Symmetry type: POINT
Atomic model buildingPDB-ID: 7P30

7p30


Accession code: 7P30 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00531866
ELECTRON MICROSCOPYf_angle_d0.62243394
ELECTRON MICROSCOPYf_dihedral_angle_d13.54795
ELECTRON MICROSCOPYf_chiral_restr0.0445030
ELECTRON MICROSCOPYf_plane_restr0.0055346

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