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- PDB-5v8f: Structural basis of MCM2-7 replicative helicase loading by ORC-Cd... -

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Entry
Database: PDB / ID: 5v8f
TitleStructural basis of MCM2-7 replicative helicase loading by ORC-Cdc6 and Cdt1
DescriptorMinichromosome maintenance protein 5
(DNA replication licensing factor ...) x 5
(Cell division ...) x 2
(Origin recognition complex subunit ...) x 6
KeywordsREPLICATION / DNA replication / Cryo-EM / OCCM
Specimen sourceSaccharomyces cerevisiae (strain atcc 204508 / s288c) / yeast / Baker's yeast /
Synthetic construct
MethodElectron microscopy (3.9 Å resolution / Particle / Single particle)
AuthorsYuan, Z. / Riera, A. / Bai, L. / Sun, J. / Spanos, C. / Chen, Z.A. / Barbon, M. / Rappsilber, J. / Stillman, B. / Speck, C. / Li, H.
CitationNat. Struct. Mol. Biol., 2017, 24, 316-324

Nat. Struct. Mol. Biol., 2017, 24, 316-324 StrPapers
Structural basis of Mcm2-7 replicative helicase loading by ORC-Cdc6 and Cdt1.
Zuanning Yuan / Alberto Riera / Lin Bai / Jingchuan Sun / Saikat Nandi / Christos Spanos / Zhuo Angel Chen / Marta Barbon / Juri Rappsilber / Bruce Stillman / Christian Speck / Huilin Li

Validation Report
SummaryFull reportAbout validation report
DateDeposition: Mar 21, 2017 / Release: May 10, 2017
RevisionDateData content typeGroupCategoryItemProviderType
1.0May 10, 2017Structure modelrepositoryInitial release
1.1Sep 20, 2017Structure modelAuthor supporting evidence / Data collectionem_image_scans / pdbx_audit_support_pdbx_audit_support.funding_organization

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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
8: Cell division cycle protein CDT1
9: Cell division control protein 6
A: Origin recognition complex subunit 1
B: Origin recognition complex subunit 2
C: Origin recognition complex subunit 3
D: Origin recognition complex subunit 4
E: Origin recognition complex subunit 5
F: Origin recognition complex subunit 6
M: DNA (39-MER)
N: DNA (39-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,170,84424
Polyers1,166,65816
Non-polymers4,1868
Water0
#1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)107750
ΔGint (kcal/M)-455
Surface area (Å2)325600
MethodPISA

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Components

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

#1: Polypeptide(L)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)
References: UniProt: P29469, EC: 3.6.4.12

Cellular component

Molecular function

Biological process

  • cellular response to DNA damage stimulus (GO: 0006974)
  • DNA strand elongation involved in DNA replication (GO: 0006271)
  • DNA unwinding involved in DNA replication (GO: 0006268)
  • double-strand break repair via break-induced replication (GO: 0000727)
  • mitotic DNA replication initiation (GO: 1902975)
  • negative regulation of ATP-dependent DNA helicase activity (GO: 1902450)
  • pre-replicative complex assembly involved in nuclear cell cycle DNA replication (GO: 0006267)
  • regulation of cell proliferation (GO: 0042127)
#2: Polypeptide(L)DNA replication licensing factor MCM3 / Minichromosome maintenance protein 3


Mass: 107653.508 Da / Num. of mol.: 1
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
References: UniProt: P24279, EC: 3.6.4.12

Cellular component

Molecular function

Biological process

  • chromatin silencing at silent mating-type cassette (GO: 0030466)
  • chromatin silencing at telomere (GO: 0006348)
  • DNA duplex unwinding (GO: 0032508)
  • DNA replication initiation (GO: 0006270)
  • DNA strand elongation involved in DNA replication (GO: 0006271)
  • double-strand break repair via break-induced replication (GO: 0000727)
  • mitotic DNA replication initiation (GO: 1902975)
  • pre-replicative complex assembly involved in nuclear cell cycle DNA replication (GO: 0006267)
#3: Polypeptide(L)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)
References: UniProt: P30665, EC: 3.6.4.12

Cellular component

Molecular function

Biological process

  • DNA replication initiation (GO: 0006270)
  • DNA strand elongation involved in DNA replication (GO: 0006271)
  • DNA unwinding involved in DNA replication (GO: 0006268)
  • double-strand break repair via break-induced replication (GO: 0000727)
  • nuclear DNA replication (GO: 0033260)
  • pre-replicative complex assembly involved in nuclear cell cycle DNA replication (GO: 0006267)
#5: Polypeptide(L)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)
References: UniProt: P53091, EC: 3.6.4.12

Cellular component

Molecular function

Biological process

  • DNA replication initiation (GO: 0006270)
  • DNA strand elongation involved in DNA replication (GO: 0006271)
  • DNA unwinding involved in DNA replication (GO: 0006268)
  • double-strand break repair via break-induced replication (GO: 0000727)
  • pre-replicative complex assembly involved in nuclear cell cycle DNA replication (GO: 0006267)
#6: Polypeptide(L)DNA replication licensing factor MCM7 / Cell division control protein 47 / Minichromosome maintenance protein 7


Mass: 90310.883 Da / Num. of mol.: 1
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
References: UniProt: P38132, EC: 3.6.4.12

Cellular component

Molecular function

Biological process

  • chromatin silencing at silent mating-type cassette (GO: 0030466)
  • chromatin silencing at telomere (GO: 0006348)
  • DNA replication initiation (GO: 0006270)
  • DNA strand elongation involved in DNA replication (GO: 0006271)
  • DNA unwinding involved in DNA replication (GO: 0006268)
  • double-strand break repair via break-induced replication (GO: 0000727)
  • nuclear DNA replication (GO: 0033260)
  • pre-replicative complex assembly involved in nuclear cell cycle DNA replication (GO: 0006267)

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Cell division ... , 2 types, 2 molecules 89

#7: Polypeptide(L)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)
References: UniProt: P47112

Cellular component

Molecular function

Biological process

  • cell division (GO: 0051301)
  • double-strand break repair via break-induced replication (GO: 0000727)
  • pre-replicative complex assembly involved in nuclear cell cycle DNA replication (GO: 0006267)
  • regulation of DNA-dependent DNA replication initiation (GO: 0030174)
#8: Polypeptide(L)Cell division control protein 6


Mass: 58112.367 Da / Num. of mol.: 1
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
References: UniProt: P09119

Cellular component

Molecular function

Biological process

  • cell division (GO: 0051301)
  • DNA replication initiation (GO: 0006270)
  • G1/S transition of mitotic cell cycle (GO: 0000082)
  • pre-replicative complex assembly involved in nuclear cell cycle DNA replication (GO: 0006267)
  • regulation of chromatin silencing at telomere (GO: 0031938)
  • regulation of DNA-dependent DNA replication initiation (GO: 0030174)

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Origin recognition complex subunit ... , 6 types, 6 molecules ABCDEF

#9: Polypeptide(L)Origin recognition complex subunit 1 / Origin recognition complex 120 kDa subunit


Mass: 104433.008 Da / Num. of mol.: 1
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
References: UniProt: P54784

Cellular component

Molecular function

Biological process

  • chromatin silencing at silent mating-type cassette (GO: 0030466)
  • DNA replication initiation (GO: 0006270)
  • pre-replicative complex assembly involved in nuclear cell cycle DNA replication (GO: 0006267)
#10: Polypeptide(L)Origin recognition complex subunit 2 / Origin recognition complex 71 kDa subunit


Mass: 71342.180 Da / Num. of mol.: 1
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
References: UniProt: P32833

Cellular component

Molecular function

Biological process

  • chromatin silencing at silent mating-type cassette (GO: 0030466)
  • chromatin silencing at telomere (GO: 0006348)
  • DNA replication initiation (GO: 0006270)
  • histone H3-K4 methylation (GO: 0051568)
  • pre-replicative complex assembly involved in nuclear cell cycle DNA replication (GO: 0006267)
#11: Polypeptide(L)Origin recognition complex subunit 3 / Origin recognition complex 62 kDa subunit


Mass: 72161.766 Da / Num. of mol.: 1
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
References: UniProt: P54790

Cellular component

Molecular function

Biological process

  • chromatin silencing at silent mating-type cassette (GO: 0030466)
  • DNA replication initiation (GO: 0006270)
  • pre-replicative complex assembly involved in nuclear cell cycle DNA replication (GO: 0006267)
#12: Polypeptide(L)Origin recognition complex subunit 4 / Origin recognition complex 56 kDa subunit


Mass: 60772.152 Da / Num. of mol.: 1
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
References: UniProt: P54791

Cellular component

Molecular function

Biological process

  • chromatin silencing at silent mating-type cassette (GO: 0030466)
  • DNA replication initiation (GO: 0006270)
  • pre-replicative complex assembly involved in nuclear cell cycle DNA replication (GO: 0006267)
#13: Polypeptide(L)Origin recognition complex subunit 5 / Origin recognition complex 53 kDa subunit


Mass: 55347.168 Da / Num. of mol.: 1
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
References: UniProt: P50874

Cellular component

Molecular function

Biological process

  • chromatin silencing at silent mating-type cassette (GO: 0030466)
  • DNA replication initiation (GO: 0006270)
  • pre-replicative complex assembly involved in nuclear cell cycle DNA replication (GO: 0006267)
  • regulation of chromatin silencing at telomere (GO: 0031938)
#14: Polypeptide(L)Origin recognition complex subunit 6 / ACS-associated protein 1 / Origin recognition complex 50 kDa subunit


Mass: 50369.531 Da / Num. of mol.: 1
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
References: UniProt: P38826

Cellular component

Molecular function

Biological process

  • chromatin silencing at silent mating-type cassette (GO: 0030466)
  • DNA replication initiation (GO: 0006270)
  • pre-replicative complex assembly involved in nuclear cell cycle DNA replication (GO: 0006267)

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DNA chain , 2 types, 2 molecules MN

#15: DNA chainDNA (39-MER)


Mass: 11967.674 Da / Num. of mol.: 1 / Source: (gene. exp.) synthetic construct
#16: DNA chainDNA (39-MER)


Mass: 12035.815 Da / Num. of mol.: 1 / Source: (gene. exp.) synthetic construct

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Polypeptide(L) / Non-polymers , 2 types, 9 molecules 5

#17: Chemical
ChemComp-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE / ATP-gamma-S (energy-carrying molecule analogue) *YM


Mass: 523.247 Da / Num. of mol.: 8 / Formula: C10H16N5O12P3S
#4: Polypeptide(L)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)
References: UniProt: P29496, EC: 3.6.4.12

Cellular component

Molecular function

Biological process

  • chromatin silencing at telomere (GO: 0006348)
  • DNA replication initiation (GO: 0006270)
  • DNA strand elongation involved in DNA replication (GO: 0006271)
  • double-strand break repair via break-induced replication (GO: 0000727)
  • establishment of chromatin silencing (GO: 0006343)
  • negative regulation of chromatin silencing at telomere (GO: 0031939)
  • nuclear DNA replication (GO: 0033260)
  • pre-replicative complex assembly involved in nuclear cell cycle DNA replication (GO: 0006267)
  • regulation of DNA-dependent DNA replication initiation (GO: 0030174)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: SINGLE PARTICLE

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Sample preparation

ComponentName: OCCM / Type: COMPLEX
Entity ID: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16
Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae
Source (recombinant)Organism: Saccharomyces cerevisiae
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 / Grid type: C-Flat
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 %

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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 FIELD / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 5 sec. / Electron dose: 10 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 304288 / Symmetry type: POINT
Atomic model buildingRef protocol: RIGID BODY FIT

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