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

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Basic information

Entry
Database: PDB / ID: 11sv
TitleStructure of Yarrowia lipolytica ORC-Cdc6 bound to 60bp segment of OriA-006 mutant CNNGGNR 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: 2.56 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)
K: Cell division control protein 6 (Cdc6)
L: Cell division control protein 6 (Cdc6)
X: DNA (60-MER)
Y: DNA (60-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)624,29919
Polymers622,17311
Non-polymers2,1268
Water28816
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 for a separate experiment, runs at expected size, with a higher ...Evidence: electron microscopy, not applicable, gel filtration, Replicates of sample were run on Superose 6 increase 3.2/300 column for a separate experiment, 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.
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.,Contained N-terminal TwinStrep-SUMOstar-TEV tag which was left on to ...Details: Contained N-terminal TwinStrep-SUMOstar-TEV tag which was left on to improve solubility of complex during concentration.,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) / YALI0C00671p


Mass: 66850.094 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: 18335.758 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Yarrowia lipolytica (yeast)
#9: DNA chain DNA (60-MER)


Mass: 18650.898 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Yarrowia lipolytica (yeast)

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Non-polymers , 3 types, 24 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.: 16 / 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 a mutant of OriA-006 containing the CNNGGNR mutation
Type: COMPLEX
Details: Purified YlORC1-6 was mixed with glycerol-free buffer containing ATP and magnesium acetate, a 60 bp mutant OriA-006 fragment, and YlCdc6 at an ORC:DNA:Cdc6 molar ratio of 1:1.5:6 in a ...Details: Purified YlORC1-6 was mixed with glycerol-free buffer containing ATP and magnesium acetate, a 60 bp mutant OriA-006 fragment, and YlCdc6 at an ORC:DNA:Cdc6 molar ratio of 1:1.5:6 in a stepwise fashion. Final protein concentration of 1.6 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
110.4895 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: 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.: 1.6 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 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 at 25C and 95% humidity, blotted for 2. ...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 at 25C and 95% humidity, blotted for 2.7 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: 2000 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 54.8 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8428
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. FYlODC60bpOri-A006-CNNGGNR data collection included 8428 exposures from a single session, with 40 frames per movie, a dose rate of 1.37 e/A^2 per frame, and a cumulative dose of 54.8 e/A^2.

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Processing

EM software
IDNameVersionCategoryDetails (eV)
1cryoSPARC4.7.1particle selectioncryoSPARC was used for template picking of particles.
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 assignment
12cryoSPARC4.7.1classificationNon-uniform refinement was carried out for the final reconstruction.
13cryoSPARC4.7.13D reconstruction
CTF correctionDetails: Patch CTF correction was carried out in cryoSPARC using the default settings, and was optimized during the refinements/reconstruction of the map in cryoSPARC.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2710398
Details: 8,428 movies were imported into cryoSPARC and underwent patch motion correction and patch CTF corrections to generate corrected micrographs. Template picking of micrographs commenced using ...Details: 8,428 movies were imported into cryoSPARC and underwent patch motion correction and patch CTF corrections to generate corrected micrographs. Template picking of micrographs commenced using representative 2D class averages of the particles used in the final YlODC60bpOri-A006-WT refinement, resulting in 4,708,382 particles being picked. Micrographs and respective particles were then analyzed using the Micrograph Junk Detector job, and after exposure and particle curation resulted in 2,710,398 particles from 7,330 micrographs. Particles were then extracted with a box size of 432 px and Fourier cropped to 128 px.
3D reconstructionResolution: 2.56 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 51222 / 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: The YlODC60bpOriA-006-WT structure was docked into the map using ChimeraX and used as a starting point, with the DNA sequence altered at the mutated sites and manual refinement in Coot used to refine the model.
Atomic model buildingDetails: The initial model came from the experimental structure of YlODC60bpOriA-006-WT
Source name: Other / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 69.93 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003925329
ELECTRON MICROSCOPYf_angle_d0.515834528
ELECTRON MICROSCOPYf_chiral_restr0.0413925
ELECTRON MICROSCOPYf_plane_restr0.00384162
ELECTRON MICROSCOPYf_dihedral_angle_d15.6614006

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