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- PDB-11su: Structure of Yarrowia lipolytica ORC-Cdc6 bound to 60bp segment o... -

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Basic information

Entry
Database: PDB / ID: 11su
TitleStructure of Yarrowia lipolytica ORC-Cdc6 bound to 60bp segment of OriA-006 DNA
Components
  • (DNA (60-MER)) x 2
  • (Origin recognition complex subunit ...) x 6
  • Cell division control protein 6 (Cdc6)
KeywordsREPLICATION / Origin Recognition Complex / ORC / origin licensing / ATPase
Function / homology
Function and homology information


nuclear DNA replication / nuclear origin of replication recognition complex / nuclear pre-replicative complex / DNA replication preinitiation complex / mitotic DNA replication checkpoint signaling / DNA replication origin binding / DNA replication initiation / DNA replication / protein-macromolecule adaptor activity / chromatin binding ...nuclear DNA replication / nuclear origin of replication recognition complex / nuclear pre-replicative complex / DNA replication preinitiation complex / mitotic DNA replication checkpoint signaling / DNA replication origin binding / DNA replication initiation / DNA replication / protein-macromolecule adaptor activity / chromatin binding / chromatin / ATP hydrolysis activity / DNA binding / ATP binding / nucleus
Similarity search - Function
Origin recognition complex, subunit 6, fungi / : / Cdc6/ORC-like, ATPase lid domain / : / Origin recognition complex subunit 1 C-terminal winged HTH domain / Orc1-like, AAA ATPase domain / AAA ATPase domain / Origin recognition complex, subunit 6 / Origin recognition complex subunit 6 (ORC6) / Cdc6, C-terminal ...Origin recognition complex, subunit 6, fungi / : / Cdc6/ORC-like, ATPase lid domain / : / Origin recognition complex subunit 1 C-terminal winged HTH domain / Orc1-like, AAA ATPase domain / AAA ATPase 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 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 / : / : / Origin recognition complex subunit 2 RecA-like domain / ORC2 WHD / Origin recognition complex, subunit 2 / : / NACHT nucleoside triphosphatase / NACHT-NTPase domain profile. / Bromo adjacent homology (BAH) domain / Bromo adjacent homology (BAH) domain superfamily / BAH domain profile. / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / 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) / YALI0F31647p / YALI0F14773p / Origin recognition complex subunit 4 / Origin recognition complex subunit 2 / Origin recognition complex subunit 1 / YALI0C00671p / YALI0B01452p
Similarity search - Component
Biological speciesYarrowia lipolytica (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.64 Å
AuthorsBauer, J. / Joshua-Tor, L.
Funding support United States, 1items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: To Be Published
Title: Evolution of Origin Sequence and Recognition for Licensing of Eukaryotic DNA Replication
Authors: Bauer, J. / Zali, N. / Chouhan, O.P. / El Demerdash, O. / Loell, K. / Kinney, J. / Joshua-Tor, L. / Stillman, B.
History
DepositionMar 11, 2026Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 30, 2026Provider: repository / Type: Initial release
Revision 1.0Sep 30, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: 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 (Orc1)
B: Origin recognition complex subunit 2 (Orc2)
C: Origin recognition complex subunit 3 (Orc3)
D: Origin recognition complex subunit 4 (Orc4)
E: Origin recognition complex subunit 5 (Orc5)
F: Origin recognition complex subunit 6 (Orc6)
G: Cell division control protein 6 (Cdc6)
X: DNA (60-MER)
Y: DNA (60-MER)
K: Cell division control protein 6 (Cdc6)
L: Cell division control protein 6 (Cdc6)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)624,47019
Polymers622,34411
Non-polymers2,1268
Water99155
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable, gel filtration, Replicates of sample were run on Superose 6 increase 3.2/300 column, runs at expected size, with a higher 260/280 than ORC or Cdc6 ...Evidence: electron microscopy, not applicable, gel filtration, Replicates of sample were run on Superose 6 increase 3.2/300 column, runs at expected size, with a higher 260/280 than ORC or Cdc6 alone, indicating bound DNA. SDS-PAGE of sample clearly displays all ORC proteins in addition to Cdc6 co-eluting., light scattering, Mass photometry analysis indicated the presence of a complex estimated to be 488 plus/minus 35 kDa, near the expected size of 489 kDa.
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 (Orc1)


Mass: 96564.922 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Contained N-terminal TwinStrep-SUMOstar-TEV tag which was left on to improve solubility of complex during concentration.
Source: (gene. exp.) Yarrowia lipolytica (yeast) / Gene: YALI0_D10104g / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q6C9L7
#2: Protein Origin recognition complex subunit 2 (Orc2)


Mass: 57479.059 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Yarrowia lipolytica (yeast) / Gene: YALI0_D22330g / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q6C865
#3: Protein Origin recognition complex subunit 3 (Orc3) / YALI0F14773p


Mass: 78749.531 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Yarrowia lipolytica (yeast) / Gene: YALI0_F14773g / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q6C1N4
#4: Protein Origin recognition complex subunit 4 (Orc4)


Mass: 57684.289 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Yarrowia lipolytica (yeast) / Gene: YALI0_E15928g / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q6C5R0
#5: Protein Origin recognition complex subunit 5 (Orc5) / YALI0B01452p


Mass: 52939.547 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Yarrowia lipolytica (yeast) / Gene: YALI0_B01452g / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q6CG25
#6: Protein Origin recognition complex subunit 6 (Orc6) / YALI0F31647p


Mass: 41218.355 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Yarrowia lipolytica (yeast) / Gene: YALI0_F31647g / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q6BZQ7

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Protein , 1 types, 3 molecules GKL

#7: Protein Cell division control protein 6 (Cdc6)


Mass: 66907.148 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Details: Originally contained an N-terminal 8xHis-TEV tag for purification, which was subsequently cleaved using TEV protease. Chains K and L are N-terminal regions of Cdc6.
Source: (gene. exp.) Yarrowia lipolytica (yeast) / Gene: YALI0_C00671g / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): CodonPlus (DE3)-RIPL / References: UniProt: Q6CDG7

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DNA chain , 2 types, 2 molecules XY

#8: DNA chain DNA (60-MER)


Mass: 18255.709 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Yarrowia lipolytica (yeast)
#9: DNA chain DNA (60-MER)


Mass: 18730.945 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Yarrowia lipolytica (yeast)

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Non-polymers , 3 types, 63 molecules

#10: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg
#11: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#12: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 55 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN
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: ORC-Cdc6 complex of Yarrowia lipolytica bound to a 60bp DNA segment of OriA-006
Type: COMPLEX
Details: Purified YlORC1-6 was mixed with glycerol-free buffer containing ATP and magnesium acetate, a 60 bp OriA-006 fragment, and YlCdc6 at an ORC:DNA:Cdc6 molar ratio of 1:1.5:4 in a stepwise ...Details: Purified YlORC1-6 was mixed with glycerol-free buffer containing ATP and magnesium acetate, a 60 bp OriA-006 fragment, and YlCdc6 at an ORC:DNA:Cdc6 molar ratio of 1:1.5:4 in a stepwise fashion. Final protein concentration of 1 mg/mL, with 0.05% lauryl maltose neopentyl glycol (LMNG). Blotted using a Leica EM GP2 automatic plunge freezer.
Entity ID: #1-#9 / Source: RECOMBINANT
Molecular weightValue: 0.4895 MDa / Experimental value: NO
Source (natural)Organism: Yarrowia lipolytica (yeast)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Plasmid: DH10 MultiBac
Buffer solutionpH: 7.5
Details: 50 mM HEPES pH 7.5, 150 mM KOAc, 10 mM Mg(OAc)2, 1 mM ATP, 1 mM DTT
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPES1
2150 mMpotassium acetateKOAc1
310 mMmagnesium acetateMgOAc1
41 mMadenosine triphosphateATP1
51 mMdithiothreitolDTT1
60.05 %lauryl maltose neopentyl glycol1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Pre-incubated in assembly buffer (50 mM HEPES pH 7.5, 150 mM KOAc, 10 mM Mg(OAc)2, 1 mM ATP, 1 mM DTT) for 10 minutes.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: EMS Lacey Carbon
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 298 K
Details: Sample was applied to a glow-discharged lacey carbon grid and blotted for 3.0 seconds, and plunged into liquid ethane using a Leica Automatic Plunge Freezer EM GP2.

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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 magnification: 105000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 78.8 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8340
Details: Cryo-electron microscopy data were collected using an FEI/ThermoFisher Titan Krios TEM operating at 300 keV. A Gatan K3 direct electron detector equipped with a BioQuantum energy filter was ...Details: Cryo-electron microscopy data were collected using an FEI/ThermoFisher Titan Krios TEM operating at 300 keV. A Gatan K3 direct electron detector equipped with a BioQuantum energy filter was utilized to semi-automatically collect dose-fractionated movies with ThermoFisher EPU data collection software. For the YlODC60bpOri-A006-WT data collection, a single session was used to collect 8340 exposures, with movies containing 40 frames at a dose rate of 1.97 e/A^2 per frame, totaling 78.8 e/A^2 in cumulative dose.

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Processing

EM software
IDNameVersionCategoryDetails (eV)
1Warpparticle selection
2PHENIX2.0_5885model refinement
5cryoSPARC4.7.1CTF correctioncryoSPARC Patch CTF Refinement and final CTF refinement in the Non-Uniform refinement job provided final CTF estimation/corrections.
10cryoSPARC4.7.1initial Euler assignmentcryoSPARC heterogeneous refinement was used for initial angular assignment
11cryoSPARC4.7.1final Euler assignmentNon-uniform refinement was carried out for the final angle assignment.
12cryoSPARC4.7.1classificationNon-uniform refinement was carried out for the final 3D classification.
13cryoSPARC4.7.13D reconstructionNon-uniform refinement was carried out for the final reconstruction.
CTF correctionDetails: CTF correction was done first in WARP during initial screening, and re-corrected in cryoSPARC during the re-picking of particles from micrographs and during the refinements/reconstruction of the map in cryoSPARC
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 793509
Details: Particle picking used a BoxNet pre-trained neural network implemented in TensorFlow, with a particle diameter of 180 angstrom and a threshold score of 0.4
3D reconstructionResolution: 2.64 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 125267 / Algorithm: FOURIER SPACE
Details: Non-uniform refinement was used for the final reconstruction.
Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: The YlODC54bpOriC-061 structure was docked into the map using ChimeraX and used as a starting point, with the DNA sequences substituted and manual refinement in Coot used to build the model.
Atomic model buildingDetails: The initial model came from the experimental structure of YlODC54bpOriC-061
Source name: Other / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 56.64 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.012325539
ELECTRON MICROSCOPYf_angle_d0.752234836
ELECTRON MICROSCOPYf_chiral_restr0.05793956
ELECTRON MICROSCOPYf_plane_restr0.00594184
ELECTRON MICROSCOPYf_dihedral_angle_d16.43284077

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