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

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Basic information

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 CDT1
  • Minichromosome maintenance protein 5
KeywordsHYDROLASE / helicase / DNA replication
Function / homologyDNA replication factor Cdt1, C-terminal / DNA replication factor Cdt1 C-terminal domain / MCM core complex / negative regulation of ATP-dependent DNA helicase activity / MCM complex binding / Switching of origins to a post-replicative state / rt:r-sce-69300: / THO complex / CMG complex / nuclear DNA replication ...DNA replication factor Cdt1, C-terminal / DNA replication factor Cdt1 C-terminal domain / MCM core complex / negative regulation of ATP-dependent DNA helicase activity / MCM complex binding / Switching of origins to a post-replicative state / rt:r-sce-69300: / THO complex / CMG complex / nuclear DNA replication / DNA replication licensing factor Mcm5 / DNA replication licensing factor Mcm3 / DNA replication licensing factor Mcm7 / DNA replication licensing factor Mcm6 / Mini-chromosome maintenance complex protein 4 / mitotic DNA replication initiation / DNA replication licensing factor Mcm2 / negative regulation of chromatin silencing at telomere / Mini-chromosome maintenance, conserved site / MCM family signature. / MCM complex / Activation of the pre-replicative complex / establishment of chromatin silencing / Mini-chromosome maintenance protein 2 / nuclear pre-replicative complex / DNA replication preinitiation complex / regulation of DNA-dependent DNA replication initiation / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / MCM N-terminal domain / MCM OB domain / MCM family domain profile. / Mini-chromosome maintenance protein / MCM domain / single-stranded DNA-dependent ATPase activity / double-strand break repair via break-induced replication / nuclear replication fork / DNA strand elongation involved in DNA replication / replication fork protection complex / DNA unwinding involved in DNA replication / MCM N-terminal domain / DNA duplex unwinding / MCM2/3/5 family / chromatin silencing at silent mating-type cassette / MCM OB domain / DNA helicase activity / chromatin silencing at telomere / DNA replication origin binding / DNA replication initiation / Orc1 removal from chromatin / DNA helicase / helicase activity / chromosome, telomeric region / single-stranded DNA binding / nuclear chromosome, telomeric region / AAA+ ATPase domain / Nucleic acid-binding, OB-fold / cell division / cellular response to DNA damage stimulus / chromatin binding / P-loop containing nucleoside triphosphate hydrolase / nucleoplasm / ATP binding / nucleus / metal ion binding / cytosol / cytoplasm / 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
Function and homology information
Specimen sourceSaccharomyces cerevisiae / Yeast / yeast /
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 7.1 Å resolution
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

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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
Polyers677,9767
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: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Strain: ATCC 204508 / S288c / Gene: MCM2, YBL023C, YBL0438 / Production host: Saccharomyces cerevisiae / References: UniProt:P29469, EC:3.6.4.12 (DNA helicase)
#2: Protein/peptide DNA replication licensing factor MCM3 / Minichromosome maintenance protein 3


Mass: 110774.703 Da / Num. of mol.: 1
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Strain: ATCC 204508 / S288c / Gene: MCM3, YEL032W, SYGP-ORF23 / Production host: Saccharomyces cerevisiae / References: UniProt:P24279, EC:3.6.4.12 (DNA helicase)
#3: Protein/peptide DNA replication licensing factor MCM4 / Cell division control protein 54


Mass: 105138.375 Da / Num. of mol.: 1
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Strain: ATCC 204508 / S288c / Gene: MCM4, CDC54, HCD21, YPR019W, YP9531.13 / Production host: Saccharomyces cerevisiae / References: UniProt:P30665, EC:3.6.4.12 (DNA helicase)
#5: Protein/peptide DNA replication licensing factor MCM6 / Minichromosome maintenance protein 6


Mass: 113110.211 Da / Num. of mol.: 1
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Strain: ATCC 204508 / S288c / Gene: MCM6, YGL201C / Production host: Saccharomyces cerevisiae / References: UniProt:P53091, EC:3.6.4.12 (DNA helicase)
#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: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Strain: ATCC 204508 / S288c / Gene: MCM7, CDC47, YBR202W, YBR1441 / Production host: Saccharomyces cerevisiae / References: UniProt:P38132, EC:3.6.4.12 (DNA helicase)

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


Mass: 68486.055 Da / Num. of mol.: 1
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Strain: ATCC 204508 / S288c / Gene: TAH11, CDT1, SID2, YJR046W, J1641 / Production host: Saccharomyces cerevisiae / References: UniProt:P47112

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 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
Source (recombinant)Organism: Saccharomyces cerevisiae
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
MicroscopyMicroscope model: 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 / Number of particles: 63000 / Symmetry type: POINT

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