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- PDB-9i3i: Cryo-EM structure of the MCM-ORC (MO) complex featuring an ORC2 r... -

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Basic information

Entry
Database: PDB / ID: 9i3i
TitleCryo-EM structure of the MCM-ORC (MO) complex featuring an ORC2 regulatory domain involved in cell cycle regulation of MCM-DH loading for DNA replication.
Components
  • (DNA (88-MER)) x 2
  • (DNA replication licensing factor ...) x 5
  • (Origin recognition complex subunit ...) x 6
  • Minichromosome maintenance protein 5
KeywordsREPLICATION / DNA Replication / Origin licensing / MCM2-7 helicase / Origin Recognition Complex / CDK / cell cycle
Function / homology
Function and homology information


CDC6 association with the ORC:origin complex / Cul8-RING ubiquitin ligase complex / maintenance of rDNA / MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / MCM complex binding / nuclear DNA replication / premeiotic DNA replication / Assembly of the ORC complex at the origin of replication ...CDC6 association with the ORC:origin complex / Cul8-RING ubiquitin ligase complex / maintenance of rDNA / MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / MCM complex binding / nuclear DNA replication / premeiotic DNA replication / Assembly of the ORC complex at the origin of replication / replication fork protection complex / nuclear origin of replication recognition complex / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / mitotic DNA replication / Activation of the pre-replicative complex / CMG complex / nuclear pre-replicative complex / nucleosome organization / Activation of ATR in response to replication stress / DNA replication preinitiation complex / MCM complex / mitotic DNA replication checkpoint signaling / double-strand break repair via break-induced replication / mitotic DNA replication initiation / single-stranded DNA helicase activity / regulation of DNA-templated DNA replication initiation / silent mating-type cassette heterochromatin formation / DNA strand elongation involved in DNA replication / Orc1 removal from chromatin / nuclear replication fork / regulation of DNA replication / DNA replication origin binding / DNA replication initiation / subtelomeric heterochromatin formation / nucleosome binding / DNA helicase activity / transcription elongation by RNA polymerase II / helicase activity / heterochromatin formation / single-stranded DNA binding / DNA helicase / chromosome, telomeric region / DNA replication / DNA damage response / chromatin binding / ATP hydrolysis activity / zinc ion binding / nucleoplasm / ATP binding / metal ion binding / nucleus / cytoplasm
Similarity search - Function
Origin recognition complex, subunit 6, fungi / : / Origin recognition complex subunit 1 C-terminal winged HTH domain / Origin recognition complex, subunit 6 / Origin recognition complex subunit 6 (ORC6) / Origin recognition complex subunit 4 / Origin recognition complex, subunit 3 / Origin recognition complex, subunit 5 / Origin recognition complex subunit 4, C-terminal / Origin recognition complex subunit 3, winged helix C-terminal ...Origin recognition complex, subunit 6, fungi / : / Origin recognition complex subunit 1 C-terminal winged HTH domain / Origin recognition complex, subunit 6 / Origin recognition complex subunit 6 (ORC6) / Origin recognition complex subunit 4 / Origin recognition complex, subunit 3 / Origin recognition complex, subunit 5 / Origin recognition complex subunit 4, C-terminal / Origin recognition complex subunit 3, winged helix C-terminal / Origin recognition complex subunit 3, N-terminal / : / : / Origin recognition complex (ORC) subunit 3 N-terminus / Origin recognition complex (ORC) subunit 4 C-terminus / Origin recognition complex (ORC) subunit 5 C-terminus / Origin recognition complex winged helix C-terminal / ORC5, lid domain / Orc1-like, AAA ATPase domain / : / : / Origin recognition complex subunit 2 RecA-like domain / ORC2 WHD / AAA ATPase domain / Origin recognition complex, subunit 2 / AAA lid domain / AAA lid domain / : / : / MCM3 winged helix domain / : / MCM5, C-terminal domain / DNA replication licensing factor MCM7, winged helix / DNA replication licensing factor Mcm5 / MCM4, winged helix domain / DNA replication licensing factor Mcm3 / Mini-chromosome maintenance complex protein 4 / : / MCM3-like, winged helix domain / DNA replication licensing factor Mcm6 / DNA replication licensing factor Mcm7 / Mcm6, C-terminal winged-helix domain / MCM6 C-terminal winged-helix domain / DNA replication licensing factor Mcm2 / Mini-chromosome maintenance protein 2 / Mini-chromosome maintenance, conserved site / MCM family signature. / Bromo adjacent homology domain / BAH domain / Bromo adjacent homology (BAH) domain / Bromo adjacent homology (BAH) domain superfamily / BAH domain profile. / MCM N-terminal domain / MCM N-terminal domain / MCM OB domain / MCM OB domain / Mini-chromosome maintenance protein / MCM, AAA-lid domain / MCM P-loop domain / MCM AAA-lid domain / MCM family domain profile. / minichromosome maintenance proteins / MCM domain / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / EF-Hand 1, calcium-binding site / Winged helix-like DNA-binding domain superfamily / Nucleic acid-binding, OB-fold / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / DNA replication licensing factor MCM3 / DNA replication licensing factor MCM2 / Minichromosome maintenance protein 5 / DNA replication licensing factor MCM4 / Origin recognition complex subunit 2 / DNA replication licensing factor MCM7 ...ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / DNA replication licensing factor MCM3 / DNA replication licensing factor MCM2 / Minichromosome maintenance protein 5 / DNA replication licensing factor MCM4 / Origin recognition complex subunit 2 / DNA replication licensing factor MCM7 / Origin recognition complex subunit 6 / Origin recognition complex subunit 5 / DNA replication licensing factor MCM6 / Origin recognition complex subunit 1 / Origin recognition complex subunit 3 / Origin recognition complex subunit 4
Similarity search - Component
Biological speciesSaccharomyces cerevisiae S288C (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsMiller, T.C.R. / Lim, C.T. / Diffley, J.F.X. / Costa, A.
Funding support United Kingdom, European Union, Denmark, 6items
OrganizationGrant numberCountry
Wellcome TrustFC0010065 United Kingdom
Medical Research Council (MRC, United Kingdom)FC0010065 United Kingdom
Cancer Research UKFC0010065 United Kingdom
European Research Council (ERC)820102European Union
Novo Nordisk FoundationNNF22OC0073571 Denmark
The Carlsberg FoundationCF21_0571 Denmark
Citation
Journal: Nat Struct Mol Biol / Year: 2025
Title: Cell cycle regulation has shaped replication origins in budding yeast.
Authors: Chew Theng Lim / Thomas C R Miller / Kang Wei Tan / Saurabh Talele / Anne Early / Philip East / Humberto Sánchez / Nynke H Dekker / Alessandro Costa / John F X Diffley /
Abstract: Eukaryotic DNA replication initiates from genomic loci known as origins. At budding yeast origins like ARS1, a double hexamer (DH) of the MCM replicative helicase is assembled by origin recognition ...Eukaryotic DNA replication initiates from genomic loci known as origins. At budding yeast origins like ARS1, a double hexamer (DH) of the MCM replicative helicase is assembled by origin recognition complex (ORC), Cdc6 and Cdt1 by sequential hexamer loading from two opposed ORC binding sites. Cyclin-dependent kinase (CDK) inhibits DH assembly, which prevents re-replication by restricting helicase loading to the G1 phase. Here, we show that an intrinsically disordered region (IDR) in the Orc2 subunit promotes interaction between ORC and the first loaded, closed-ring MCM hexamer (the MCM-ORC (MO) intermediate). CDK-dependent phosphorylation of this IDR blocks MO formation and DH assembly. We show that MO stabilizes ORC at lower-affinity binding sites required for second hexamer loading. Origins comprising two high-affinity ORC sites can assemble DH efficiently without MO by independently loading single hexamers. Strikingly, these origins escape CDK inhibition in vitro and in vivo. Our work reveals mechanistic plasticity in MCM loading with implications for understanding how CDK regulation has shaped yeast origin evolution and how natural, strong origins might escape cell cycle regulation. We also identify key steps common to loading pathways, with implications for understanding how MCM is loaded in other eukaryotes.
#1: Journal: Nature / Year: 2019
Title: Mechanism of head-to-head MCM double-hexamer formation revealed by cryo-EM.
Authors: Thomas C R Miller / Julia Locke / Julia F Greiwe / John F X Diffley / Alessandro Costa /
Abstract: In preparation for bidirectional DNA replication, the origin recognition complex (ORC) loads two hexameric MCM helicases to form a head-to-head double hexamer around DNA. The mechanism of MCM double- ...In preparation for bidirectional DNA replication, the origin recognition complex (ORC) loads two hexameric MCM helicases to form a head-to-head double hexamer around DNA. The mechanism of MCM double-hexamer formation is debated. Single-molecule experiments have suggested a sequential mechanism, in which the ORC-dependent loading of the first hexamer drives the recruitment of the second hexamer. By contrast, biochemical data have shown that two rings are loaded independently via the same ORC-mediated mechanism, at two inverted DNA sites. Here we visualize MCM loading using time-resolved electron microscopy, and identify intermediates in the formation of the double hexamer. We confirm that both hexamers are recruited via the same interaction that occurs between ORC and the C-terminal domains of the MCM helicases. Moreover, we identify the mechanism of coupled MCM loading. The loading of the first MCM hexamer around DNA creates a distinct interaction site, which promotes the engagement of ORC at the N-terminal homodimerization interface of MCM. In this configuration, ORC is poised to direct the recruitment of the second hexamer in an inverted orientation, which is suitable for the formation of the double hexamer. Our results therefore reconcile the two apparently contrasting models derived from single-molecule experiments and biochemical data.
History
DepositionJan 23, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 25, 2025Provider: repository / Type: Initial release
Revision 1.1Jul 16, 2025Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
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
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
X: DNA (88-MER)
Y: DNA (88-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,086,94329
Polymers1,083,31314
Non-polymers3,63015
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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

#1: Protein Origin recognition complex subunit 1 / Origin recognition complex 120 kDa subunit


Mass: 108612.922 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: N-terminus contains a CBP-TEV purification tag. / Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: ORC1, YML065W / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P54784
#2: Protein Origin recognition complex subunit 2 / Origin recognition complex 71 kDa subunit


Mass: 71342.180 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: ORC2, RRR1, SIR5, YBR060C, YBR0523 / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P32833
#3: Protein Origin recognition complex subunit 3 / Origin recognition complex 62 kDa subunit


Mass: 72161.766 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: ORC3, OAF1, OIF1, YLL004W, L1365 / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P54790
#4: Protein Origin recognition complex subunit 4 / Origin recognition complex 56 kDa subunit


Mass: 60772.152 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: ORC4, YPR162C, P9325.5 / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P54791
#5: Protein Origin recognition complex subunit 5 / Origin recognition complex 53 kDa subunit


Mass: 55347.168 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: ORC5, YNL261W, N0834 / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P50874
#6: Protein Origin recognition complex subunit 6 / ACS-associated protein 1 / Origin recognition complex 50 kDa subunit


Mass: 50369.531 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: ORC6, AAP1, YHR118C / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P38826

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

#7: Protein 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 S288C (yeast) / Gene: MCM2, YBL023C, YBL0438 / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P29469, DNA helicase
#8: Protein DNA replication licensing factor MCM3 / Minichromosome maintenance protein 3


Mass: 111720.242 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: N-terminus contains a CBP-TEV purification tag / Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: MCM3, YEL032W, SYGP-ORF23 / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P24279, DNA helicase
#9: Protein 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 S288C (yeast) / Gene: MCM4, CDC54, HCD21, YPR019W, YP9531.13 / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P30665, DNA helicase
#11: Protein 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 S288C (yeast) / Gene: MCM6, YGL201C / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P53091, DNA helicase
#12: Protein 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 S288C (yeast) / Gene: MCM7, CDC47, YBR202W, YBR1441 / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P38132, DNA helicase

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Protein , 1 types, 1 molecules 5

#10: Protein 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 S288C (yeast) / Gene: MCM5, CDC46, YLR274W, L9328.1 / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P29496, DNA helicase

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DNA chain , 2 types, 2 molecules XY

#13: DNA chain DNA (88-MER)


Mass: 27116.334 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae S288C (yeast)
#14: DNA chain DNA (88-MER)


Mass: 27154.562 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae S288C (yeast)

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Non-polymers , 4 types, 15 molecules

#15: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#16: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#17: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#18: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
Has protein modificationN

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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: MCM-ORC featuring ORC2 regulatory domain / Type: COMPLEX / Entity ID: #1-#14 / Source: RECOMBINANT
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae S288C (yeast)
Buffer solutionpH: 7.6
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 288 K
Details: 10 second incubation, 3.5 seconds single side blotting.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 4200 nm / Nominal defocus min: 2700 nm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50.4 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1

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Processing

EM software
IDNameVersionCategory
1RELION3particle selection
4Gctf1.18CTF correction
7Cootmodel fitting
9RELION3initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 6287507
3D reconstructionResolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 177687 / Symmetry type: POINT

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