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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
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/homologyOrigin recognition complex, subunit 6, fungi / Assembly of the ORC complex at the origin of replication / rt:r-sce-69298: / CDC6 association with the ORC:origin complex / Origin recognition complex, subunit 6 / Origin recognition complex subunit 6 (ORC6) / Cell division protein Cdc6/18 / Origin recognition complex subunit 4 / Origin recognition complex subunit 4, C-terminal / Origin recognition complex, subunit 3 ...Origin recognition complex, subunit 6, fungi / Assembly of the ORC complex at the origin of replication / rt:r-sce-69298: / CDC6 association with the ORC:origin complex / Origin recognition complex, subunit 6 / Origin recognition complex subunit 6 (ORC6) / Cell division protein Cdc6/18 / Origin recognition complex subunit 4 / Origin recognition complex subunit 4, C-terminal / Origin recognition complex, subunit 3 / Origin recognition complex, subunit 5 / Origin recognition complex, subunit 2 / Origin recognition complex (ORC) subunit 5 C-terminus / Origin recognition complex (ORC) subunit 4 C-terminus / Origin recognition complex subunit 2 / DNA replication factor Cdt1, C-terminal / Cdc6, C-terminal / AAA domain / DNA replication factor Cdt1 C-terminal domain / nuclear origin of replication recognition complex / Origin recognition complex, subunit 1 / Switching of origins to a post-replicative state / rt:r-sce-69300: / negative regulation of ATP-dependent DNA helicase activity / MCM core complex / THO complex / CMG complex / nuclear DNA replication / regulation of chromatin silencing at telomere / CDC6, C terminal winged helix domain / DNA replication licensing factor Mcm7 / DNA replication licensing factor Mcm / DNA replication licensing factor Mcm3 / Mini-chromosome maintenance complex protein 4 / DNA replication licensing factor Mcm6 / 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 / establishment of chromatin silencing / Activation of the pre-replicative complex / Mini-chromosome maintenance protein 2 / DNA replication preinitiation complex / nuclear pre-replicative 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 domain / MCM family domain profile. / Mini-chromosome maintenance protein / single-stranded DNA-dependent ATPase activity / double-strand break repair via break-induced replication / nuclear replication fork / BAH domain profile. / Bromo adjacent homology (BAH) domain / DNA strand elongation involved in DNA replication / replication fork protection complex / DNA unwinding involved in DNA replication / BAH domain / DNA duplex unwinding / MCM N-terminal domain / histone H3-K4 methylation / 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 / CDK-mediated phosphorylation and removal of Cdc6 / Orc1 removal from chromatin / DNA helicase / ATPase, AAA-type, core / helicase activity / chromosome, telomeric region / single-stranded DNA binding / ATPase family associated with various cellular activities (AAA) / G1/S transition of mitotic cell cycle / chromosome / nuclear chromosome, telomeric region / ATPase activity / AAA+ ATPase domain / regulation of cell proliferation / Winged helix DNA-binding domain superfamily / Nucleic acid-binding, OB-fold / GTPase activity / Winged helix-like DNA-binding domain superfamily / cell division / GTP binding / cellular response to DNA damage stimulus / chromatin binding / P-loop containing nucleoside triphosphate hydrolase / nucleoplasm / ATP binding / nucleus / metal ion binding / cytosol
Function and homology information
Specimen sourceSaccharomyces cerevisiae / yeast / /
Synthetic construct
MethodElectron microscopy (3.9 Å resolution / Particle / Single particle) / Transmission electron microscopy
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
Abstract: To initiate DNA replication, the origin recognition complex (ORC) and Cdc6 load an Mcm2-7 double hexamer onto DNA. Without ATP hydrolysis, ORC-Cdc6 recruits one Cdt1-bound Mcm2-7 hexamer, thus ...To initiate DNA replication, the origin recognition complex (ORC) and Cdc6 load an Mcm2-7 double hexamer onto DNA. Without ATP hydrolysis, ORC-Cdc6 recruits one Cdt1-bound Mcm2-7 hexamer, thus forming an ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) helicase-loading intermediate. Here we report a 3.9-Å structure of Saccharomyces cerevisiae OCCM on DNA. Flexible Mcm2-7 winged-helix domains (WHDs) engage ORC-Cdc6. A three-domain Cdt1 configuration embraces Mcm2, Mcm4, and Mcm6, thus comprising nearly half of the hexamer. The Cdt1 C-terminal domain extends to the Mcm6 WHD, which binds the Orc4 WHD. DNA passes through the ORC-Cdc6 and Mcm2-7 rings. Origin DNA interaction is mediated by an α-helix within Orc4 and positively charged loops within Orc2 and Cdc6. The Mcm2-7 C-tier AAA+ ring is topologically closed by an Mcm5 loop that embraces Mcm2, but the N-tier-ring Mcm2-Mcm5 interface remains open. This structure suggests a loading mechanism of the first Cdt1-bound Mcm2-7 hexamer by ORC-Cdc6.
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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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)
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: 107653.508 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: 90310.883 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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Cell division ... , 2 types, 2 molecules 89

#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
#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)
Strain: ATCC 204508 / S288c / Gene: CDC6, YJL194W, J0347 / Production host: Saccharomyces cerevisiae / 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)
Strain: ATCC 204508 / S288c / Gene: ORC1, YML065W / Production host: Saccharomyces cerevisiae / 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)
Strain: ATCC 204508 / S288c / Gene: ORC2, RRR1, SIR5, YBR060C, YBR0523 / Production host: Saccharomyces cerevisiae / 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)
Strain: ATCC 204508 / S288c / Gene: ORC3, OAF1, OIF1, YLL004W, L1365 / Production host: Saccharomyces cerevisiae / 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)
Strain: ATCC 204508 / S288c / Gene: ORC4, YPR162C, P9325.5 / Production host: Saccharomyces cerevisiae / 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)
Strain: ATCC 204508 / S288c / Gene: ORC5, YNL261W, N0834 / Production host: Saccharomyces cerevisiae / 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)
Strain: ATCC 204508 / S288c / Gene: ORC6, AAP1, YHR118C / Production host: Saccharomyces cerevisiae / 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 / Production host: synthetic construct
#16: DNA chain DNA (39-MER)


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

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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)
Strain: ATCC 204508 / S288c / Gene: MCM5, CDC46, YLR274W, L9328.1 / Production host: Saccharomyces cerevisiae / References: UniProt:P29496, EC:3.6.4.12 (DNA helicase)

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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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