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- PDB-11rl: CryoEM structure of the human origin recognition complex with DNA... -

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Basic information

Entry
Database: PDB / ID: 11rl
TitleCryoEM structure of the human origin recognition complex with DNA and CDC6 protein
Components
  • (DNA (29-MER)) x 2
  • (Origin recognition complex subunit ...) x 5
  • Cell division control protein 6 homolog
KeywordsREPLICATION / Origin Recognition Complex / ORC / origin licensing / WH domain / ATPase / protein-DNA complex
Function / homology
Function and homology information


cellular response to vasopressin / traversing start control point of mitotic cell cycle / positive regulation of chromosome segregation / CDC6 association with the ORC:origin complex / polar body extrusion after meiotic divisions / origin recognition complex / E2F-enabled inhibition of pre-replication complex formation / inner kinetochore / nuclear origin of replication recognition complex / nuclear pre-replicative complex ...cellular response to vasopressin / traversing start control point of mitotic cell cycle / positive regulation of chromosome segregation / CDC6 association with the ORC:origin complex / polar body extrusion after meiotic divisions / origin recognition complex / E2F-enabled inhibition of pre-replication complex formation / inner kinetochore / nuclear origin of replication recognition complex / nuclear pre-replicative complex / DNA replication checkpoint signaling / DNA replication preinitiation complex / negative regulation of DNA replication / regulation of cyclin-dependent protein serine/threonine kinase activity / mitotic DNA replication checkpoint signaling / Transcription of E2F targets under negative control by DREAM complex / regulation of mitotic metaphase/anaphase transition / G1/S-Specific Transcription / spindle midzone / positive regulation of cytokinesis / cellular response to angiotensin / regulation of DNA replication / DNA replication origin binding / Activation of the pre-replicative complex / DNA replication initiation / protein polymerization / Activation of ATR in response to replication stress / heterochromatin / positive regulation of fibroblast proliferation / Assembly of the ORC complex at the origin of replication / protein serine/threonine kinase binding / Assembly of the pre-replicative complex / CDK-mediated phosphorylation and removal of Cdc6 / Orc1 removal from chromatin / spindle pole / DNA replication / cell division / chromosome, telomeric region / protein-macromolecule adaptor activity / chromosome / nucleotide binding / negative regulation of cell population proliferation / centrosome / chromatin binding / nucleolus / chromatin / negative regulation of transcription by RNA polymerase II / ATP hydrolysis activity / DNA binding / nucleoplasm / ATP binding / membrane / nucleus / cytosol / cytoplasm
Similarity search - Function
Cell division protein Cdc6/18 / Origin recognition complex subunit 3, insertion domain / Origin recognition complex subunit 3 insertion domain / : / Cdc6/ORC-like, ATPase lid domain / CDC6, C terminal / Orc1-like, AAA ATPase domain / AAA ATPase domain / Cdc6, C-terminal / CDC6, C terminal winged helix domain ...Cell division protein Cdc6/18 / Origin recognition complex subunit 3, insertion domain / Origin recognition complex subunit 3 insertion domain / : / Cdc6/ORC-like, ATPase lid domain / CDC6, C terminal / Orc1-like, AAA ATPase domain / AAA ATPase domain / 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 / AAA lid domain / AAA lid domain / : / Bromo adjacent homology domain / BAH domain / 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 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) / Origin recognition complex subunit 5 / Origin recognition complex subunit 4 / Origin recognition complex subunit 1 / Origin recognition complex subunit 2 / DNA replication factor CDC6 / Origin recognition complex subunit 3
Similarity search - Component
Biological speciesHomo sapiens (human)
DNA molecule (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsChouhan, O.P. / 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 10, 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
B: Origin recognition complex subunit 2
D: Origin recognition complex subunit 4
H: DNA (29-MER)
I: DNA (29-MER)
A: Origin recognition complex subunit 1
C: Origin recognition complex subunit 3
E: Origin recognition complex subunit 5
G: Cell division control protein 6 homolog
hetero molecules


Theoretical massNumber of molelcules
Total (without water)448,83516
Polymers446,7098
Non-polymers2,1268
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Origin recognition complex subunit ... , 5 types, 5 molecules BDACE

#1: Protein Origin recognition complex subunit 2


Mass: 66063.375 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ORC2, ORC2L / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q13416
#2: Protein Origin recognition complex subunit 4


Mass: 50443.266 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: No Tag, Native protein / Source: (gene. exp.) Homo sapiens (human) / Gene: ORC4, ORC4L / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O43929
#5: Protein Origin recognition complex subunit 1 / Replication control protein 1


Mass: 97556.914 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: N-terminal TwinStrep-SUMOstar-TEV tag, which was cleaved off using TEV protease, and G remained at the N-terminal after TEV cleavage.
Source: (gene. exp.) Homo sapiens (human) / Gene: ORC1, ORC1L, PARC1 / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q13415
#6: Protein Origin recognition complex subunit 3 / Origin recognition complex subunit Latheo


Mass: 82422.109 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: N-terminal TwinStrep-SUMOstar-TEV tag, which was cleaved off using TEV protease, and G remained at the N-terminal after TEV cleavage.
Source: (gene. exp.) Homo sapiens (human) / Gene: ORC3, LATHEO, ORC3L / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9UBD5
#7: Protein Origin recognition complex subunit 5


Mass: 50349.934 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ORC5, ORC5L / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O43913

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DNA chain , 2 types, 2 molecules HI

#3: DNA chain DNA (29-MER)


Mass: 18630.834 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) DNA molecule (others)
#4: DNA chain DNA (29-MER)


Mass: 18364.727 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) DNA molecule (others)

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Protein , 1 types, 1 molecules G

#8: Protein Cell division control protein 6 homolog / CDC6-related protein / Cdc18-related protein / HsCdc18 / p62(cdc6) / HsCDC6


Mass: 62877.402 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: N-terminal 8XHis-SUMOstar-TEV tag, which was cleaved off using TEV protease, and G remained at the N-terminal after TEV cleavage.
Source: (gene. exp.) Homo sapiens (human) / Gene: CDC6, CDC18L / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Rosetta / References: UniProt: Q99741

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Non-polymers , 2 types, 8 molecules

#9: Chemical
ChemComp-ATP / ADENOSINE-5'-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
#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

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Details

Has ligand of interestY
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: Human ORC subunits (1-5) and Human CDC6 protein complex with 60bp DNA
Type: COMPLEX
Details: Purified proteins were mixed with 60bp DNA in glycerol-free buffer containing ATP analogs and magnesium acetate. ORC:CDC6:DNA molar ratio of 1:2:3. Final protein concentration of 1 mg/mL, ...Details: Purified proteins were mixed with 60bp DNA in glycerol-free buffer containing ATP analogs and magnesium acetate. ORC:CDC6:DNA molar ratio of 1:2:3. 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-#8 / Source: RECOMBINANT
Molecular weightValue: 0.408 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Plasmid: DH10 MultiBac
Buffer solutionpH: 7.5
Details: 25 mM HEPES pH 7.5, 100 mM KCl, 2 mM DTT, 0.05% LMNG
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMPottesium chlorideKCl1
225 mMHEPESC8H18N2O4S1
32 mMDTTC4H10O2S21
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 KCl, 5 mM Mg(OAc)2, 2 mM DTT) for 20 minutes
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 298 K
Details: The sample was applied to a glow-discharged Quantifoil R 0.6/1 300 mesh copper grid (previously washed with ethyl acetate), incubated for 10 seconds, blotted for 3 seconds, and plunged into ...Details: The sample was applied to a glow-discharged Quantifoil R 0.6/1 300 mesh copper grid (previously washed with ethyl acetate), incubated for 10 seconds, blotted for 3 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
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: 2 / Num. of real images: 7088
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, ...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 using ThermoFisher EPU data collection software.

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Processing

EM software
IDNameVersionCategoryDetails (eV)
1Warp1.0.9particle selectionBoxNet functionality was used to pick particles.
2cryoSPARC4.7.1particle selectioncryoSPARC was used for 2D classification and sorting of WARP-picked particle sets.
3Warp1.0.9image acquisitionWARP was utilized for motion correction, CTF estimation, micrograph generation, particle picking, and particle stack generation
4EPUimage acquisitionEPU was used for semi-automatic collection of exposures from chosen grid squares.
6Warp1.0.9CTF correctionWARP did inital CTF estimation and correction to whole micrographs and particle stacks
9Coot0.9.8.95model fitting
11cryoSPARC4.7.1initial Euler assignmentcryoSPARC heterogeneous refinement was used for initial angular assignment
12cryoSPARC4.7.1final Euler assignment
13cryoSPARC4.7.1classificationNon-uniform refinement was carried out for the final reconstruction.
14cryoSPARC4.7.13D reconstruction
15PHENIX1.21.2_5419model refinement
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 selectionDetails: 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: 2.8 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 130819 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: Initial local fitting was done using Chimera, and then Coot was used for flexible fitting and model building.
Atomic model buildingPDB-ID: 7JPS

7jps


Accession code: 7JPS / Source name: PDB / Type: experimental model
RefinementHighest resolution: 2.8 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00221804
ELECTRON MICROSCOPYf_angle_d0.46129777
ELECTRON MICROSCOPYf_dihedral_angle_d17.2413419
ELECTRON MICROSCOPYf_chiral_restr0.0373432
ELECTRON MICROSCOPYf_plane_restr0.0043561

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