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- PDB-9bcx: Cryo-EM structure of the S. cerevisiae ORC-Cdc6-Mcm2-7-DNA comple... -

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Basic information

Entry
Database: PDB / ID: 9bcx
TitleCryo-EM structure of the S. cerevisiae ORC-Cdc6-Mcm2-7-DNA complex with a fully closed Mcm2-Mcm5 DNA entry gate
Components
  • (DNA (39-MER)) x 2
  • (DNA replication licensing factor ...) x 6
  • (Origin recognition complex subunit ...) x 6
  • Cell division control protein 6
  • TAH11 isoform 1
KeywordsDNA BINDING PROTEIN / DNA replication / Cryo-EM / OCCM-deltaC6 / REPLICATION-DNA complex
Function / homology
Function and homology information


MCM complex loading / : / 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 ...MCM complex loading / : / 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 / Activation of the pre-replicative complex / mitotic DNA replication / CMG complex / nuclear pre-replicative complex / cyclin-dependent protein serine/threonine kinase inhibitor activity / nucleosome organization / DNA replication preinitiation complex / Activation of ATR in response to replication stress / MCM complex / mitotic DNA replication checkpoint signaling / double-strand break repair via break-induced replication / mitotic DNA replication initiation / single-stranded DNA helicase activity / silent mating-type cassette heterochromatin formation / regulation of DNA-templated DNA replication initiation / DNA strand elongation involved in DNA replication / CDK-mediated phosphorylation and removal of Cdc6 / 3'-5' DNA helicase activity / Orc1 removal from chromatin / nuclear replication fork / regulation of DNA replication / DNA replication origin binding / DNA replication initiation / subtelomeric heterochromatin formation / nucleosome binding / G1/S transition of mitotic cell cycle / helicase activity / single-stranded DNA binding / chromosome / DNA helicase / chromosome, telomeric region / DNA replication / hydrolase activity / cell division / GTPase activity / DNA damage response / chromatin binding / GTP binding / ATP hydrolysis activity / zinc ion binding / nucleoplasm / ATP binding / metal ion binding / nucleus / cytoplasm
Similarity search - Function
Origin recognition complex, subunit 6, fungi / Cell division protein Cdc6/18 / : / Cdc6/ORC-like, ATPase lid domain / : / Origin recognition complex subunit 1 C-terminal winged HTH domain / Origin recognition complex, subunit 6 / Origin recognition complex subunit 6 (ORC6) / Cdc6, C-terminal / CDC6, C terminal winged helix domain ...Origin recognition complex, subunit 6, fungi / Cell division protein Cdc6/18 / : / Cdc6/ORC-like, ATPase lid domain / : / Origin recognition complex subunit 1 C-terminal winged HTH domain / Origin recognition complex, subunit 6 / Origin recognition complex subunit 6 (ORC6) / Cdc6, C-terminal / CDC6, C terminal winged helix domain / : / AAA domain / 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 / : / DNA replication licensing factor MCM7, winged helix / DNA replication licensing factor Mcm5 / 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 / MCM N-terminal domain / MCM N-terminal domain / Bromo adjacent homology (BAH) domain / Bromo adjacent homology (BAH) domain superfamily / BAH domain profile. / 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 DNA-binding domain superfamily / 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'-TRIPHOSPHATE / DNA / DNA (> 10) / DNA replication licensing factor MCM5 / : / : / : / Cell division control protein 6 / DNA replication licensing factor MCM2 / Origin recognition complex subunit 2 ...ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / DNA replication licensing factor MCM5 / : / : / : / Cell division control protein 6 / DNA replication licensing factor MCM2 / 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 (brewer's yeast)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.1 Å
AuthorsYuan, Z. / Bai, L. / Li, H. / Speck, C.
Funding support United States, United Kingdom, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM110387 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM131754 United States
Biotechnology and Biological Sciences Research Council (BBSRC)BB/S001387/1 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/N000323/1 United Kingdom
Wellcome Trust107903/Z/15/Z United Kingdom
CitationJournal: Nat Commun / Year: 2025
Title: MCM2-7 ring closure involves the Mcm5 C-terminus and triggers Mcm4 ATP hydrolysis.
Authors: Sarah V Faull / Marta Barbon / Audrey Mossler / Zuanning Yuan / Lin Bai / L Maximilian Reuter / Alberto Riera / Christian Winkler / Indiana Magdalou / Matthew Peach / Huilin Li / Christian Speck /
Abstract: The eukaryotic helicase MCM2-7, is loaded by ORC, Cdc6 and Cdt1 as a double-hexamer onto replication origins. The insertion of DNA into the helicase leads to partial MCM2-7 ring closure, while ATP ...The eukaryotic helicase MCM2-7, is loaded by ORC, Cdc6 and Cdt1 as a double-hexamer onto replication origins. The insertion of DNA into the helicase leads to partial MCM2-7 ring closure, while ATP hydrolysis is essential for consecutive steps in pre-replicative complex (pre-RC) assembly. Currently it is unknown how MCM2-7 ring closure and ATP-hydrolysis are controlled. A cryo-EM structure of an ORC-Cdc6-Cdt1-MCM2-7 intermediate shows a remodelled, fully-closed Mcm2/Mcm5 interface. The Mcm5 C-terminus (C5) contacts Orc3 and specifically recognises this closed ring. Interestingly, we found that normal helicase loading triggers Mcm4 ATP-hydrolysis, which in turn leads to reorganisation of the MCM2-7 complex and Cdt1 release. However, defective MCM2-7 ring closure, due to mutations at the Mcm2/Mcm5 interface, leads to MCM2-7 ring splitting and complex disassembly. As such we identify Mcm4 as the key ATPase in regulating pre-RC formation. Crucially, a stable Mcm2/Mcm5 interface is essential for productive ATP-hydrolysis-dependent remodelling of the helicase.
History
DepositionApr 10, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 1, 2025Provider: repository / Type: Initial release
Revision 1.1Jan 15, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
2: DNA replication licensing factor MCM2
3: DNA replication licensing factor MCM3
5: DNA replication licensing factor MCM5
4: DNA replication licensing factor MCM4
6: DNA replication licensing factor MCM6
7: DNA replication licensing factor MCM7
B: Origin recognition complex subunit 1
C: Origin recognition complex subunit 2
D: Origin recognition complex subunit 3
E: Origin recognition complex subunit 4
F: Origin recognition complex subunit 5
G: Origin recognition complex subunit 6
I: Cell division control protein 6
8: TAH11 isoform 1
O: DNA (39-MER)
P: DNA (39-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,173,12923
Polymers1,171,51016
Non-polymers1,6197
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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DNA replication licensing factor ... , 6 types, 6 molecules 235467

#1: 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 (brewer's yeast)
Gene: MCM2, YBL023C, YBL0438 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P29469, DNA helicase
#2: Protein DNA replication licensing factor MCM3


Mass: 107653.508 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: MCM3, GI527_G0001749 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A8H8ULI2, DNA helicase
#3: Protein DNA replication licensing factor MCM5


Mass: 86505.734 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: MCM5, GI527_G0004088 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A6A5PUY8, DNA helicase
#4: Protein DNA replication licensing factor MCM4


Mass: 105138.375 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: MCM4, GI527_G0006071 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A8H4BU27, DNA helicase
#5: 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 (brewer's yeast)
Gene: MCM6, YGL201C / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P53091, DNA helicase
#6: 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 (brewer's yeast)
Gene: MCM7, CDC47, YBR202W, YBR1441 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P38132, DNA helicase

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

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


Mass: 104546.164 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: ORC1 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P54784
#8: 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 (brewer's yeast)
Gene: ORC2 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P32833
#9: 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 (brewer's yeast)
Gene: ORC / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P54790
#10: 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 (brewer's yeast)
Gene: ORC4, YPR162C, P9325.5 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P54791
#11: 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 (brewer's yeast)
Gene: ORC5, YNL261W, N0834 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P50874
#12: 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 (brewer's yeast)
Gene: ORC6, AAP1, YHR118C / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P38826

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Protein , 2 types, 2 molecules I8

#13: Protein Cell division control protein 6


Mass: 58112.367 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: CDC6, YJL194W, J0347 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P09119
#14: Protein TAH11 isoform 1


Mass: 68486.055 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: TAH11, GI527_G0003298 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A8H4C095

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DNA chain , 2 types, 2 molecules OP

#15: DNA chain DNA (39-MER)


Mass: 11967.674 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#16: DNA chain DNA (39-MER)


Mass: 12035.815 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 2 types, 7 molecules

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


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


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg

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Details

Has ligand of interestN
Has protein modificationY

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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: ORC-Cdc6-Mcm2-7-dsDNA / Type: COMPLEX / Entity ID: #1-#16 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast) / Plasmid: pRS
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 6.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 53903 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT

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