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- PDB-7mca: Structure of the S. cerevisiae origin recognition complex bound t... -

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Basic information

Entry
Database: PDB / ID: 7mca
TitleStructure of the S. cerevisiae origin recognition complex bound to the replication initiator Cdc6 and the ARS1 origin DNA.
Components
  • (DNA (85-MER)) x 2
  • (Origin recognition complex subunit ...) x 6
  • Cell division control protein 6
KeywordsREPLICATION / replication initiation
Function / homology
Function and homology information


CDK-mediated phosphorylation and removal of Cdc6 / CDC6 association with the ORC:origin complex / Assembly of the ORC complex at the origin of replication / Orc1 removal from chromatin / Cul8-RING ubiquitin ligase complex / maintenance of rDNA / nuclear origin of replication recognition complex / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / Activation of the pre-replicative complex / nuclear pre-replicative complex ...CDK-mediated phosphorylation and removal of Cdc6 / CDC6 association with the ORC:origin complex / Assembly of the ORC complex at the origin of replication / Orc1 removal from chromatin / Cul8-RING ubiquitin ligase complex / maintenance of rDNA / nuclear origin of replication recognition complex / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / Activation of the pre-replicative complex / nuclear pre-replicative complex / Activation of ATR in response to replication stress / DNA replication preinitiation complex / mitotic DNA replication checkpoint signaling / silent mating-type cassette heterochromatin formation / regulation of DNA-templated DNA replication initiation / DNA replication origin binding / regulation of DNA replication / subtelomeric heterochromatin formation / DNA replication initiation / nucleosome binding / G1/S transition of mitotic cell cycle / chromosome / chromosome, telomeric region / cell division / GTPase activity / chromatin binding / GTP binding / ATP hydrolysis activity / nucleoplasm / ATP binding / metal ion binding / nucleus / cytoplasm
Similarity search - Function
Origin recognition complex, subunit 6, fungi / Cell division protein Cdc6/18 / : / Origin recognition complex subunit 1 C-terminal winged HTH domain / Origin recognition complex, subunit 6 / Origin recognition complex subunit 6 (ORC6) / AAA domain / Origin recognition complex subunit 4 / Origin recognition complex, subunit 3 / Origin recognition complex, subunit 5 ...Origin recognition complex, subunit 6, fungi / Cell division protein Cdc6/18 / : / Origin recognition complex subunit 1 C-terminal winged HTH domain / Origin recognition complex, subunit 6 / Origin recognition complex subunit 6 (ORC6) / 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 (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 / AAA ATPase domain / Origin recognition complex subunit 2 / Origin recognition complex, subunit 2 / AAA lid domain / AAA lid domain / Bromo adjacent homology (BAH) domain superfamily / Bromo adjacent homology domain / Bromo adjacent homology (BAH) domain / BAH domain / BAH domain profile. / 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 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / : / DNA / DNA (> 10) / Cell division control protein 6 / Origin recognition complex subunit 2 / 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: 3.6 Å
AuthorsFeng, X. / Li, H.
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM131754 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM45436 United States
Biotechnology and Biological Sciences Research Council (BBSRC)BB/S001387.1 United States
Biotechnology and Biological Sciences Research Council (BBSRC)BB/N000323/1 United States
CitationJournal: Nat Commun / Year: 2021
Title: The structure of ORC-Cdc6 on an origin DNA reveals the mechanism of ORC activation by the replication initiator Cdc6.
Authors: Xiang Feng / Yasunori Noguchi / Marta Barbon / Bruce Stillman / Christian Speck / Huilin Li /
Abstract: The Origin Recognition Complex (ORC) binds to sites in chromosomes to specify the location of origins of DNA replication. The S. cerevisiae ORC binds to specific DNA sequences throughout the cell ...The Origin Recognition Complex (ORC) binds to sites in chromosomes to specify the location of origins of DNA replication. The S. cerevisiae ORC binds to specific DNA sequences throughout the cell cycle but becomes active only when it binds to the replication initiator Cdc6. It has been unclear at the molecular level how Cdc6 activates ORC, converting it to an active recruiter of the Mcm2-7 hexamer, the core of the replicative helicase. Here we report the cryo-EM structure at 3.3 Å resolution of the yeast ORC-Cdc6 bound to an 85-bp ARS1 origin DNA. The structure reveals that Cdc6 contributes to origin DNA recognition via its winged helix domain (WHD) and its initiator-specific motif. Cdc6 binding rearranges a short α-helix in the Orc1 AAA+ domain and the Orc2 WHD, leading to the activation of the Cdc6 ATPase and the formation of the three sites for the recruitment of Mcm2-7, none of which are present in ORC alone. The results illuminate the molecular mechanism of a critical biochemical step in the licensing of eukaryotic replication origins.
History
DepositionApr 1, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 2, 2021Provider: repository / Type: Initial release
Revision 1.1Aug 4, 2021Group: 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.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 / _citation_author.name

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Structure visualization

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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
G: DNA (85-MER)
H: DNA (85-MER)
I: Cell division control protein 6
hetero molecules


Theoretical massNumber of molelcules
Total (without water)527,25617
Polymers525,0669
Non-polymers2,1908
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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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: 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: unidentified baculovirus / 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 (brewer's yeast)
Gene: ORC2 / Production host: unidentified baculovirus / 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 (brewer's yeast)
Gene: ORC / Production host: unidentified baculovirus / 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 (brewer's yeast)
Gene: ORC4, YPR162C, P9325.5 / Production host: unidentified baculovirus / 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 (brewer's yeast)
Gene: ORC5, YNL261W, N0834 / Production host: unidentified baculovirus / 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 (brewer's yeast)
Gene: ORC6, AAP1, YHR118C / Production host: unidentified baculovirus / References: UniProt: P38826

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DNA chain , 2 types, 2 molecules GH

#7: DNA chain DNA (85-MER)


Mass: 26184.838 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae (brewer's yeast) / References: GenBank: 1039022962
#8: DNA chain DNA (85-MER)


Mass: 26229.906 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae (brewer's yeast) / References: GenBank: 1039022962

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Protein , 1 types, 1 molecules I

#9: 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: unidentified baculovirus / References: UniProt: P09119

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Non-polymers , 2 types, 8 molecules

#10: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#11: 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

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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: Complex of origin replication complex with Cdc6 and ARS1 origin DNA
Type: COMPLEX / Entity ID: #1-#9 / Source: RECOMBINANT
Molecular weightValue: 0.47 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: unidentified baculovirus / Cell: Hi-5 cell / Plasmid: pAcUW51
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPES1
2100 mMpotassium acetateCH3CO2K1
35 mMmagnesium acetateC4H6MgO41
45 mMATP-gamma-S1
52 mMDTT1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
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 FIELDBright-field microscopy
Image recordingElectron dose: 76 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameVersionCategory
1SerialEMimage acquisition
12PHENIX1.14model fitting
19RELION3particle selection
20CTFFIND4.1.10CTF correction
21cryoSPARC2initial Euler assignment
22cryoSPARC2final Euler assignment
23RELION3classification
24RELION33D reconstruction
25PHENIX1.14model refinement
26Coot0.8.9.1model refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 177397
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 72000 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 118.5 / Protocol: OTHER / Space: REAL / Target criteria: correlation coefficient
Atomic model buildingPDB-ID: 5V8F

5v8f

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