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- PDB-6wgc: Atomic model of semi-attached mutant OCCM-DNA complex (ORC-Cdc6-C... -

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Basic information

Entry
Database: PDB / ID: 6wgc
TitleAtomic model of semi-attached mutant OCCM-DNA complex (ORC-Cdc6-Cdt1-Mcm2-7 with Mcm6 WHD truncation)
Components
  • (DNA (41-MER)) x 2
  • (DNA replication licensing factor ...) x 2
  • (Origin recognition complex subunit ...) x 6
  • Cell division control protein 6
KeywordsREPLICATION/DNA / 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 / Assembly of the ORC complex at the origin of replication / 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 / Assembly of the ORC complex at the origin of replication / nuclear DNA replication / premeiotic DNA replication / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / nuclear origin of replication recognition complex / Activation of the pre-replicative complex / nucleosome organization / CMG complex / nuclear pre-replicative complex / Activation of ATR in response to replication stress / DNA replication preinitiation complex / MCM complex / cyclin-dependent protein serine/threonine kinase inhibitor activity / replication fork protection 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 / DNA strand elongation involved in DNA replication / silent mating-type cassette heterochromatin formation / CDK-mediated phosphorylation and removal of Cdc6 / DNA unwinding involved in DNA replication / Orc1 removal from chromatin / nuclear replication fork / regulation of DNA replication / DNA replication origin binding / nucleosome binding / DNA replication initiation / subtelomeric heterochromatin formation / helicase activity / G1/S transition of mitotic cell cycle / single-stranded DNA binding / chromosome / DNA helicase / chromosome, telomeric region / cell division / GTPase activity / chromatin binding / GTP binding / ATP hydrolysis activity / nucleoplasm / ATP binding / nucleus / metal ion binding / cytoplasm
Similarity search - Function
Origin recognition complex, subunit 6, fungi / : / Origin recognition complex subunit 1 C-terminal winged HTH domain / Cell division protein Cdc6/18 / : / Cdc6/ORC-like, ATPase lid 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 6, fungi / : / Origin recognition complex subunit 1 C-terminal winged HTH domain / Cell division protein Cdc6/18 / : / Cdc6/ORC-like, ATPase lid 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 / Cdc6, C-terminal / : / CDC6, C terminal winged helix domain / Orc1-like, AAA ATPase domain / Origin recognition complex subunit 2 / AAA domain / AAA ATPase domain / Origin recognition complex, subunit 2 / AAA lid domain / AAA lid domain / : / : / MCM3 winged helix domain / DNA replication licensing factor Mcm3 / DNA replication licensing factor Mcm7 / 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 / Mini-chromosome maintenance protein / MCM, AAA-lid domain / MCM P-loop domain / MCM OB 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 / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Nucleic acid-binding, OB-fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / DNA / DNA (> 10) / Cell division control protein 6 / DNA replication licensing factor MCM3 / Origin recognition complex subunit 2 / DNA replication licensing factor MCM7 / Origin recognition complex subunit 6 / Origin recognition complex subunit 5 / Origin recognition complex subunit 1 ...PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / DNA / DNA (> 10) / Cell division control protein 6 / DNA replication licensing factor MCM3 / Origin recognition complex subunit 2 / DNA replication licensing factor MCM7 / Origin recognition complex subunit 6 / Origin recognition complex subunit 5 / Origin recognition complex subunit 1 / Origin recognition complex subunit 3 / Origin recognition complex subunit 4
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsYuan, Z. / Schneider, S. / Dodd, T. / Riera, A. / Bai, L. / Yan, C. / Magdalou, I. / Ivanov, I. / Stillman, B. / 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: Proc Natl Acad Sci U S A / Year: 2020
Title: Structural mechanism of helicase loading onto replication origin DNA by ORC-Cdc6.
Authors: Zuanning Yuan / Sarah Schneider / Thomas Dodd / Alberto Riera / Lin Bai / Chunli Yan / Indiana Magdalou / Ivaylo Ivanov / Bruce Stillman / Huilin Li / Christian Speck /
Abstract: DNA replication origins serve as sites of replicative helicase loading. In all eukaryotes, the six-subunit origin recognition complex (Orc1-6; ORC) recognizes the replication origin. During late M- ...DNA replication origins serve as sites of replicative helicase loading. In all eukaryotes, the six-subunit origin recognition complex (Orc1-6; ORC) recognizes the replication origin. During late M-phase of the cell-cycle, Cdc6 binds to ORC and the ORC-Cdc6 complex loads in a multistep reaction and, with the help of Cdt1, the core Mcm2-7 helicase onto DNA. A key intermediate is the ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) complex in which DNA has been already inserted into the central channel of Mcm2-7. Until now, it has been unclear how the origin DNA is guided by ORC-Cdc6 and inserted into the Mcm2-7 hexamer. Here, we truncated the C-terminal winged-helix-domain (WHD) of Mcm6 to slow down the loading reaction, thereby capturing two loading intermediates prior to DNA insertion in budding yeast. In "semi-attached OCCM," the Mcm3 and Mcm7 WHDs latch onto ORC-Cdc6 while the main body of the Mcm2-7 hexamer is not connected. In "pre-insertion OCCM," the main body of Mcm2-7 docks onto ORC-Cdc6, and the origin DNA is bent and positioned adjacent to the open DNA entry gate, poised for insertion, at the Mcm2-Mcm5 interface. We used molecular simulations to reveal the dynamic transition from preloading conformers to the loaded conformers in which the loading of Mcm2-7 on DNA is complete and the DNA entry gate is fully closed. Our work provides multiple molecular insights into a key event of eukaryotic DNA replication.
History
DepositionApr 5, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 15, 2020Provider: repository / Type: Initial release
Revision 1.1Jul 22, 2020Group: Structure summary / Category: struct / Item: _struct.title
Revision 1.2Jul 29, 2020Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.3Aug 12, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.4Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
9: Cell division control protein 6
A: Origin recognition complex subunit 1
B: Origin recognition complex subunit 2
C: Origin recognition complex subunit 3
E: Origin recognition complex subunit 5
D: Origin recognition complex subunit 4
F: Origin recognition complex subunit 6
3: DNA replication licensing factor MCM3
7: DNA replication licensing factor MCM7
G: DNA (41-MER)
H: DNA (41-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)702,56715
Polymers700,47411
Non-polymers2,0934
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area53350 Å2
ΔGint-278 kcal/mol
Surface area136740 Å2

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Components

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

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


Mass: 104433.008 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: ORC1, YML065W / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P54784
#3: 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, RRR1, SIR5, YBR060C, YBR0523 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P32833
#4: 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: ORC3, OAF1, OIF1, YLL004W, L1365 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P54790
#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 (brewer's yeast)
Gene: ORC5, YNL261W, N0834 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P50874
#6: 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
#7: 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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DNA replication licensing factor ... , 2 types, 2 molecules 37

#8: Protein DNA replication licensing factor MCM3 / Minichromosome maintenance protein 3


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, YEL032W, SYGP-ORF23 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P24279, DNA helicase
#9: 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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DNA chain , 2 types, 2 molecules GH

#10: DNA chain DNA (41-MER)


Mass: 12577.062 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Production host: Saccharomyces cerevisiae (brewer's yeast)
#11: DNA chain DNA (41-MER)


Mass: 12655.295 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Production host: Saccharomyces cerevisiae (brewer's yeast)

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Protein / Non-polymers , 2 types, 5 molecules 9

#12: 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.: 4 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
#1: 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

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Details

Has ligand of interestY

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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-Mcm3-Mcm7-dsDNA / Type: COMPLEX / Entity ID: #1-#11 / Source: RECOMBINANT
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
Specimen supportDetails: unspecified
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
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
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 86074 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT

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