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- PDB-11st: Structure of Yarrowia lipolytica ORC-Cdc6 bound to 54bp segment o... -

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Basic information

Entry
Database: PDB / ID: 11st
TitleStructure of Yarrowia lipolytica ORC-Cdc6 bound to 54bp segment of OriC-061 DNA
Components
  • (DNA (54-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'-DIPHOSPHATE / 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: 2.73 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
B: Origin recognition complex subunit 2
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)
K: Cell division control protein 6 (Cdc6)
X: DNA (54-MER)
Y: DNA (54-MER)
L: Cell division control protein 6 (Cdc6)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)620,65018
Polymers618,62911
Non-polymers2,0227
Water39622
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable, gel filtration, Sample was generated from gel filtration run, runs at expected size, with a higher 260/280 than ORC or Cdc6 alone, indicating bound DNA. ...Evidence: electron microscopy, not applicable, gel filtration, Sample was generated from gel filtration run, 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 487 plus/minus 35 kDa, near the expected size of 486 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


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


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
Details: Author provided reference is GenBank XP_504002.3 / 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) / YALI0C00671p


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. A leftover glycine (residue 0) can be seen in the map. Chains K and L ...Details: Originally contained an N-terminal 8xHis-TEV tag for purification, which was subsequently cleaved using TEV protease. A leftover glycine (residue 0) can be seen in the map. 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 (54-MER)


Mass: 16551.680 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Yarrowia lipolytica (yeast)
#9: DNA chain DNA (54-MER)


Mass: 16719.715 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Yarrowia lipolytica (yeast)

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Non-polymers , 4 types, 29 molecules

#10: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg
#11: 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
#12: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#13: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 22 / 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 54bp DNA segment of OriC-061
Type: COMPLEX
Details: Purified YlORC1-6 was mixed with glycerol-free buffer containing ATP and magnesium acetate, a 54 bp OriC-061 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 54 bp OriC-061 fragment, and YlCdc6 at an ORC:DNA:Cdc6 molar ratio of 1:1.5:4 in a stepwise fashion, followed by gel filtration. Final protein concentration of 1-1.25 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 weight
IDEntity assembly-IDValue (°)Experimental value
110.4858 MDaNO
210.384 MDaNO
Source (natural)Organism: Yarrowia lipolytica (yeast)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Plasmid: DH10 MultiBac
Buffer solutionpH: 7.5
Details: 25 mM HEPES pH 7.5, 100 mM NaCl, 1 mM DTT, 0.05% LMNG
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHEPES1
2100 mMsodium chlorideNaCl1
31 mMdithiothreitol1
40.05 %lauryl maltose neopentyl glycol1
SpecimenConc.: 1.2 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, 10% glycerol) for 10 minutes before size exclusion chromatography.
Specimen supportDetails: ethyl acetate wash / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 298 K
Details: Sample was applied to a non-glow discharged Quantifoil R 1.2/1.3 300 mesh copper grid (previously washed with ethyl acetate), incubated for 10 seconds, blotted for 2.9 seconds, and plunged ...Details: Sample was applied to a non-glow discharged Quantifoil R 1.2/1.3 300 mesh copper grid (previously washed with ethyl acetate), incubated for 10 seconds, blotted for 2.9 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: 43.2 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 3 / Num. of real images: 20341
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 YlODC54bpOriC-061 data collection, 30-frame movies were collected over three consecutive days, resulting in 9309, 8758, and 2274 exposures taken, respectively, at a dose rate of 1.44 e/A^2 per frame, totaling a cumulative dose of 43.2 e/A^2.

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Processing

EM software
IDNameVersionCategory
1Warpparticle selection
2PHENIX2.0_5885model refinement
13cryoSPARC4.73D reconstruction
CTF correctionDetails: CTF correction was done first in WARP during exposure/micrograph pre-processing, and re-corrected during the final refinements/reconstruction of the map in cryoSPARC
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3805196
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 0.5
3D reconstructionResolution: 2.73 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 51599 / Algorithm: FOURIER SPACE
Details: Non-uniform refinement was used for the final reconstruction.
Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: AlphaFold 2 models for each subunit were docked into the density individually using the "fit to map" functionality in ChimeraX, then refined using Coot. The density for the DNA was sharp ...Details: AlphaFold 2 models for each subunit were docked into the density individually using the "fit to map" functionality in ChimeraX, then refined using Coot. The density for the DNA was sharp enough to allow us to discern purines and pyrimidines, allowing us to produce a generic DNA-B form model of the respective DNA sequence and manually rebuild it in Coot. PHENIX Real Space Refine functionality was used to further process and finalize the structure.
Atomic model building
ID 3D fitting-IDSource nameTypeDetails (eV)
11AlphaFoldin silico model
21Otherin silico modelGeneric B-DNA used as template, generated in Coot.
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 77.93 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00325622
ELECTRON MICROSCOPYf_angle_d0.469434941
ELECTRON MICROSCOPYf_chiral_restr0.03883971
ELECTRON MICROSCOPYf_plane_restr0.00384198
ELECTRON MICROSCOPYf_dihedral_angle_d15.63484074

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