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- PDB-7tjj: S. cerevisiae ORC bound to 84 bp ARS1 DNA and Cdc6 (state 1) with... -

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Basic information

Entry
Database: PDB / ID: 7tjj
TitleS. cerevisiae ORC bound to 84 bp ARS1 DNA and Cdc6 (state 1) with docked Orc6 N-terminal domain
Components
  • (DNA, 84 bp ARS1) x 2
  • (Origin recognition complex subunit ...) x 6
  • Cell division control protein 6
KeywordsREPLICATION/DNA / initiator / helicase loader / AAA+ ATPase / REPLICATION-DNA complex
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
ADENOSINE-5'-TRIPHOSPHATE / : / 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: 2.7 Å
AuthorsSchmidt, J.M. / Yang, R. / Kumar, A. / Hunker, O. / Bleichert, F.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01-GM141313 United States
European Research Council (ERC)ERC-STG-757909 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32-GM008283 United States
CitationJournal: Nat Commun / Year: 2022
Title: A mechanism of origin licensing control through autoinhibition of S. cerevisiae ORC·DNA·Cdc6.
Authors: Jan Marten Schmidt / Ran Yang / Ashish Kumar / Olivia Hunker / Jan Seebacher / Franziska Bleichert /
Abstract: The coordinated action of multiple replicative helicase loading factors is needed for the licensing of replication origins prior to DNA replication. Binding of the Origin Recognition Complex (ORC) to ...The coordinated action of multiple replicative helicase loading factors is needed for the licensing of replication origins prior to DNA replication. Binding of the Origin Recognition Complex (ORC) to DNA initiates the ATP-dependent recruitment of Cdc6, Cdt1 and Mcm2-7 loading, but the structural details for timely ATPase site regulation and for how loading can be impeded by inhibitory signals, such as cyclin-dependent kinase phosphorylation, are unknown. Using cryo-electron microscopy, we have determined several structures of S. cerevisiae ORC·DNA·Cdc6 intermediates at 2.5-2.7 Å resolution. These structures reveal distinct ring conformations of the initiator·co-loader assembly and inactive ATPase site configurations for ORC and Cdc6. The Orc6 N-terminal domain laterally engages the ORC·Cdc6 ring in a manner that is incompatible with productive Mcm2-7 docking, while deletion of this Orc6 region alleviates the CDK-mediated inhibition of Mcm7 recruitment. Our findings support a model in which Orc6 promotes the assembly of an autoinhibited ORC·DNA·Cdc6 intermediate to block origin licensing in response to CDK phosphorylation and to avert DNA re-replication.
History
DepositionJan 16, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 5, 2022Provider: repository / Type: Initial release

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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
G: DNA, 84 bp ARS1
H: DNA, 84 bp ARS1
I: Cell division control protein 6
hetero molecules


Theoretical massNumber of molelcules
Total (without water)527,39217
Polymers525,2669
Non-polymers2,1268
Water2,882160
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: 104818.422 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: Trichoplusia ni (cabbage looper) / 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: Trichoplusia ni (cabbage looper) / 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: Trichoplusia ni (cabbage looper) / References: UniProt: P54790
#4: Protein Origin recognition complex subunit 4 / / Origin recognition complex 56 kDa subunit


Mass: 61044.414 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: Trichoplusia ni (cabbage looper) / 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: Trichoplusia ni (cabbage looper) / 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: Trichoplusia ni (cabbage looper) / References: UniProt: P38826

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DNA chain , 2 types, 2 molecules GH

#7: DNA chain DNA, 84 bp ARS1 /


Mass: 25820.561 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae (brewer's yeast) / References: GenBank: 1039022860
#8: DNA chain DNA, 84 bp ARS1 /


Mass: 25977.766 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae (brewer's yeast) / References: GenBank: 1039022860

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Protein , 1 types, 1 molecules I

#9: Protein Cell division control protein 6


Mass: 58384.625 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: Trichoplusia ni (cabbage looper) / References: UniProt: P09119

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Non-polymers , 3 types, 168 molecules

#10: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#11: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#12: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 160 / Source method: isolated from a natural source / Formula: H2O

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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 in complex with ARS1 DNA and Cdc6 / Type: COMPLEX / Entity ID: #1-#9 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Buffer solutionpH: 7.6
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / 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: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1600 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 6.6 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 88859 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00625215
ELECTRON MICROSCOPYf_angle_d0.94234421
ELECTRON MICROSCOPYf_dihedral_angle_d17.0689534
ELECTRON MICROSCOPYf_chiral_restr0.0553919
ELECTRON MICROSCOPYf_plane_restr0.0074023

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