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- PDB-5xf8: Cryo-EM structure of the Cdt1-MCM2-7 complex in AMPPNP state -

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Entry
Database: PDB / ID: 5xf8
TitleCryo-EM structure of the Cdt1-MCM2-7 complex in AMPPNP state
Components
  • (DNA replication licensing factor ...) x 5
  • Cell division cycle protein CDT1Cell cycle
  • Minichromosome maintenance protein 5
KeywordsHYDROLASE / helicase / DNA replication
Function / homology
Function and homology information


Orc1 removal from chromatin / Activation of the pre-replicative complex / Switching of origins to a post-replicative state / MCM complex binding / go:1902450: / MCM core complex / mitotic DNA replication / CMG complex / nuclear DNA replication / negative regulation of chromatin silencing at telomere ...Orc1 removal from chromatin / Activation of the pre-replicative complex / Switching of origins to a post-replicative state / MCM complex binding / go:1902450: / MCM core complex / mitotic DNA replication / CMG complex / nuclear DNA replication / negative regulation of chromatin silencing at telomere / establishment of chromatin silencing / MCM complex / DNA replication preinitiation complex / regulation of DNA-dependent DNA replication initiation / mitotic DNA replication initiation / nuclear pre-replicative complex / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / go:0043142: / double-strand break repair via break-induced replication / nuclear replication fork / replication fork protection complex / DNA strand elongation involved in DNA replication / chromatin silencing at silent mating-type cassette / DNA duplex unwinding / DNA helicase activity / DNA unwinding involved in DNA replication / DNA replication origin binding / chromatin silencing at telomere / DNA replication initiation / ec:3.6.4.12: / helicase activity / chromosome, telomeric region / single-stranded DNA binding / nuclear chromosome, telomeric region / cell division / cellular response to DNA damage stimulus / chromatin binding / nucleoplasm / ATP binding / metal ion binding / nucleus / cytoplasm
MCM OB domain / Mini-chromosome maintenance, conserved site / Mcm6, C-terminal winged-helix domain / MCM domain / AAA+ ATPase domain / DNA replication licensing factor Mcm2 / DNA replication licensing factor Mcm3 / Mini-chromosome maintenance protein 2 / Mini-chromosome maintenance complex protein 4 / DNA replication licensing factor Mcm5 ...MCM OB domain / Mini-chromosome maintenance, conserved site / Mcm6, C-terminal winged-helix domain / MCM domain / AAA+ ATPase domain / DNA replication licensing factor Mcm2 / DNA replication licensing factor Mcm3 / Mini-chromosome maintenance protein 2 / Mini-chromosome maintenance complex protein 4 / DNA replication licensing factor Mcm5 / DNA replication licensing factor Mcm6 / DNA replication licensing factor Mcm7 / MCM P-loop domain / Nucleic acid-binding, OB-fold / MCM N-terminal domain / P-loop containing nucleoside triphosphate hydrolase / MCM OB domain / MCM AAA-lid domain / MCM6 C-terminal winged-helix domain / MCM family signature. / MCM family domain profile. / MCM N-terminal domain / Mini-chromosome maintenance protein / MCM, AAA-lid domain
DNA replication licensing factor MCM3 / DNA replication licensing factor MCM2 / Minichromosome maintenance protein 5 / DNA replication licensing factor MCM4 / DNA replication licensing factor MCM7 / Cell division cycle protein CDT1 / DNA replication licensing factor MCM6
Biological speciesSaccharomyces cerevisiae (baker's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.1 Å
AuthorsZhai, Y. / Cheng, E. / Wu, H. / Li, N. / Yung, P.Y. / Gao, N. / Tye, B.K.
CitationJournal: Nat. Struct. Mol. Biol. / Year: 2017
Title: Open-ringed structure of the Cdt1-Mcm2-7 complex as a precursor of the MCM double hexamer.
Authors: Yuanliang Zhai / Erchao Cheng / Hao Wu / Ningning Li / Philip Yuk Kwong Yung / Ning Gao / Bik-Kwoon Tye /
Abstract: The minichromosome maintenance complex (MCM) hexameric complex (Mcm2-7) forms the core of the eukaryotic replicative helicase. During G1 phase, two Cdt1-Mcm2-7 heptamers are loaded onto each ...The minichromosome maintenance complex (MCM) hexameric complex (Mcm2-7) forms the core of the eukaryotic replicative helicase. During G1 phase, two Cdt1-Mcm2-7 heptamers are loaded onto each replication origin by the origin-recognition complex (ORC) and Cdc6 to form an inactive MCM double hexamer (DH), but the detailed loading mechanism remains unclear. Here we examine the structures of the yeast MCM hexamer and Cdt1-MCM heptamer from Saccharomyces cerevisiae. Both complexes form left-handed coil structures with a 10-15-Å gap between Mcm5 and Mcm2, and a central channel that is occluded by the C-terminal domain winged-helix motif of Mcm5. Cdt1 wraps around the N-terminal regions of Mcm2, Mcm6 and Mcm4 to stabilize the whole complex. The intrinsic coiled structures of the precursors provide insights into the DH formation, and suggest a spring-action model for the MCM during the initial origin melting and the subsequent DNA unwinding.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Apr 9, 2017 / Release: May 3, 2017
RevisionDateData content typeProviderType
1.0May 3, 2017Structure modelrepositoryInitial release

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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: Minichromosome maintenance protein 5
6: DNA replication licensing factor MCM6
7: DNA replication licensing factor MCM7
C: Cell division cycle protein CDT1


Theoretical massNumber of molelcules
Total (without water)677,9767
Polymers677,9767
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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

#1: Protein/peptide 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 (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: MCM2, YBL023C, YBL0438 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P29469, EC: 3.6.4.12
#2: Protein/peptide DNA replication licensing factor MCM3 / Minichromosome maintenance protein 3


Mass: 110774.703 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: MCM3, YEL032W, SYGP-ORF23 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P24279, EC: 3.6.4.12
#3: Protein/peptide 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 (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: MCM4, CDC54, HCD21, YPR019W, YP9531.13 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P30665, EC: 3.6.4.12
#5: Protein/peptide 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 (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: MCM6, YGL201C / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P53091, EC: 3.6.4.12
#6: Protein/peptide 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 (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: MCM7, CDC47, YBR202W, YBR1441 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P38132, EC: 3.6.4.12

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Protein/peptide , 2 types, 2 molecules 5C

#4: Protein/peptide 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 (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: MCM5, CDC46, YLR274W, L9328.1 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P29496, EC: 3.6.4.12
#7: Protein/peptide Cell division cycle protein CDT1 / Cell cycle / SIC1 indispensable protein 2 / Topoisomerase-A hypersensitive protein 11


Mass: 68486.055 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: TAH11, CDT1, SID2, YJR046W, J1641 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P47112

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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: Cdt1-Mcm2-7 / Type: COMPLEX / Entity ID: 1, 2, 3, 4, 5, 6, 7 / Source: RECOMBINANT
Source (natural)Organism: Saccharomyces cerevisiae (baker's yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (baker's yeast)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER
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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 22 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 7.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 63000 / Symmetry type: POINT

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