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

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Basic information

Entry
Database: PDB / ID: 5v8f
TitleStructural basis of MCM2-7 replicative helicase loading by ORC-Cdc6 and Cdt1
Components
  • (Cell division ...) x 2
  • (DNA (39-MER)) x 2
  • (DNA replication licensing factor ...) x 5
  • (Origin recognition complex subunit ...) x 6
  • Minichromosome maintenance protein 5
KeywordsREPLICATION / DNA replication / Cryo-EM / OCCM
Function / homologyDNA replication licensing factor Mcm7 / Mini-chromosome maintenance complex protein 4 / Cdc6, C-terminal / Nucleic acid-binding, OB-fold / Origin recognition complex, subunit 6 / Mini-chromosome maintenance protein 2 / DNA replication licensing factor Mcm6 / DNA replication licensing factor Mcm5 / DNA replication licensing factor Mcm3 / Origin recognition complex subunit 4 ...DNA replication licensing factor Mcm7 / Mini-chromosome maintenance complex protein 4 / Cdc6, C-terminal / Nucleic acid-binding, OB-fold / Origin recognition complex, subunit 6 / Mini-chromosome maintenance protein 2 / DNA replication licensing factor Mcm6 / DNA replication licensing factor Mcm5 / DNA replication licensing factor Mcm3 / Origin recognition complex subunit 4 / DNA replication licensing factor Mcm2 / Origin recognition complex, subunit 2 / ATPase, AAA-type, core / AAA+ ATPase domain / MCM domain / Bromo adjacent homology (BAH) domain / CDC6, C terminal winged helix domain / Cell division protein Cdc6/18 / Origin recognition complex, subunit 6, fungi / MCM N-terminal domain / MCM OB domain / Origin recognition complex subunit 2 / BAH domain / MCM2/3/5 family / ATPase family associated with various cellular activities (AAA) / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily / Origin recognition complex subunit 4, C-terminal / Mini-chromosome maintenance, conserved site / DNA replication factor Cdt1, C-terminal / Mini-chromosome maintenance protein / MCM N-terminal domain / P-loop containing nucleoside triphosphate hydrolase / Origin recognition complex, subunit 5 / Origin recognition complex, subunit 3 / Origin recognition complex, subunit 1 / AAA domain / Origin recognition complex subunit 6 (ORC6) / Switching of origins to a post-replicative state / Origin recognition complex (ORC) subunit 4 C-terminus / Activation of the pre-replicative complex / Orc1 removal from chromatin / CDC6 association with the ORC:origin complex / Assembly of the ORC complex at the origin of replication / BAH domain profile. / MCM family domain profile. / MCM family signature. / MCM OB domain / DNA replication factor Cdt1 C-terminal domain / CDK-mediated phosphorylation and removal of Cdc6 / Origin recognition complex (ORC) subunit 5 C-terminus / rt:r-sce-69298: / nuclear origin of replication recognition complex / MCM core complex / negative regulation of ATP-dependent DNA helicase activity / MCM complex binding / regulation of chromatin silencing at telomere / CMG complex / nuclear DNA replication / mitotic DNA replication initiation / negative regulation of chromatin silencing at telomere / MCM complex / establishment of chromatin silencing / 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 / 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 / DNA duplex unwinding / histone H3-K4 methylation / chromatin silencing at silent mating-type cassette / DNA helicase activity / DNA replication origin binding / chromatin silencing at telomere / DNA replication initiation / DNA helicase / helicase activity / chromosome, telomeric region / single-stranded DNA binding / G1/S transition of mitotic cell cycle / chromosome / nuclear chromosome, telomeric region / ATPase activity / GTPase activity / cell division / GTP binding / cellular response to DNA damage stimulus / chromatin binding / nucleoplasm / ATP binding / nucleus / metal ion binding / cytoplasm / Origin recognition complex subunit 3 / Origin recognition complex subunit 6
Function and homology information
Specimen sourceSaccharomyces cerevisiae (baker's yeast)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.9 Å resolution
AuthorsYuan, Z. / Riera, A. / Bai, L. / Sun, J. / Spanos, C. / Chen, Z.A. / Barbon, M. / Rappsilber, J. / Stillman, B. / Speck, C. / Li, H.
CitationJournal: Nat. Struct. Mol. Biol. / Year: 2017
Title: Structural basis of Mcm2-7 replicative helicase loading by ORC-Cdc6 and Cdt1.
Authors: 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
1.2Nov 8, 2017Structure modelDerived calculations / Experimental preparationem_sample_support / pdbx_struct_assembly_em_sample_support.grid_type / _pdbx_struct_assembly.details / _pdbx_struct_assembly.method_details

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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
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

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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) (yeast)
Strain: ATCC 204508 / S288c / Gene: MCM2, YBL023C, YBL0438 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P29469, DNA helicase
#2: Protein/peptide 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) (yeast)
Strain: ATCC 204508 / S288c / Gene: MCM3, YEL032W, SYGP-ORF23 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P24279, 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) (yeast)
Strain: ATCC 204508 / S288c / Gene: MCM4, CDC54, HCD21, YPR019W, YP9531.13 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P30665, 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) (yeast)
Strain: ATCC 204508 / S288c / Gene: MCM6, YGL201C / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P53091, DNA helicase
#6: Protein/peptide 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) (yeast)
Strain: ATCC 204508 / S288c / Gene: MCM7, CDC47, YBR202W, YBR1441 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P38132, DNA helicase

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

#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: (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
#8: Protein/peptide Cell division control protein 6


Mass: 58112.367 Da / Num. of mol.: 1
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: CDC6, YJL194W, J0347 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P09119

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

#9: Protein/peptide 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) (yeast)
Strain: ATCC 204508 / S288c / Gene: ORC1, YML065W / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P54784
#10: Protein/peptide 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) (yeast)
Strain: ATCC 204508 / S288c / Gene: ORC2, RRR1, SIR5, YBR060C, YBR0523 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P32833
#11: Protein/peptide 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) (yeast)
Strain: ATCC 204508 / S288c / Gene: ORC3, OAF1, OIF1, YLL004W, L1365 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P54790
#12: Protein/peptide 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) (yeast)
Strain: ATCC 204508 / S288c / Gene: ORC4, YPR162C, P9325.5 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P54791
#13: Protein/peptide 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) (yeast)
Strain: ATCC 204508 / S288c / Gene: ORC5, YNL261W, N0834 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P50874
#14: Protein/peptide 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) (yeast)
Strain: ATCC 204508 / S288c / Gene: ORC6, AAP1, YHR118C / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P38826

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DNA chain , 2 types, 2 molecules MN

#15: DNA chain DNA (39-MER)


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


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

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Protein/peptide / 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


Mass: 523.247 Da / Num. of mol.: 8 / Formula: C10H16N5O12P3S
Comment: ATP-gamma-S (energy-carrying molecule analogue) *YM
#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) (yeast)
Strain: ATCC 204508 / S288c / Gene: MCM5, CDC46, YLR274W, L9328.1 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P29496, DNA helicase

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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: 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 (baker's yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (baker's yeast)
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 FIELDBright-field microscopy / 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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