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- PDB-9eo4: Outward-open structure of human dopamine transporter bound to cocaine -

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Basic information

Entry
Database: PDB / ID: 9eo4
TitleOutward-open structure of human dopamine transporter bound to cocaine
ComponentsSodium-dependent dopamine transporter
KeywordsMEMBRANE PROTEIN / neurotransmitter/sodium symporters / monoamine transporter / dopamine transporter / SLC6
Function / homology
Function and homology information


Defective SLC6A3 causes Parkinsonism-dystonia infantile (PKDYS) / Defective SLC6A3 causes Parkinsonism-dystonia infantile (PKDYS) / Dopamine clearance from the synaptic cleft / amine binding / adenohypophysis development / dopamine uptake / hyaloid vascular plexus regression / dopamine binding / dopamine:sodium symporter activity / norepinephrine:sodium symporter activity ...Defective SLC6A3 causes Parkinsonism-dystonia infantile (PKDYS) / Defective SLC6A3 causes Parkinsonism-dystonia infantile (PKDYS) / Dopamine clearance from the synaptic cleft / amine binding / adenohypophysis development / dopamine uptake / hyaloid vascular plexus regression / dopamine binding / dopamine:sodium symporter activity / norepinephrine:sodium symporter activity / norepinephrine transport / neurotransmitter transmembrane transporter activity / regulation of dopamine metabolic process / dopaminergic synapse / dopamine transport / monoamine transmembrane transporter activity / Na+/Cl- dependent neurotransmitter transporters / monoamine transport / flotillin complex / dopamine catabolic process / positive regulation of multicellular organism growth / neurotransmitter transport / response to iron ion / dopamine biosynthetic process / dopamine uptake involved in synaptic transmission / amino acid transport / heterocyclic compound binding / neuronal cell body membrane / sodium ion transmembrane transport / prepulse inhibition / axon terminus / response to cAMP / lactation / response to cocaine / protein phosphatase 2A binding / locomotory behavior / response to nicotine / cognition / sensory perception of smell / presynaptic membrane / postsynaptic membrane / response to ethanol / protease binding / response to xenobiotic stimulus / neuron projection / membrane raft / axon / signaling receptor binding / neuronal cell body / protein-containing complex binding / cell surface / membrane / metal ion binding / plasma membrane / cytoplasm
Similarity search - Function
Sodium:neurotransmitter symporter, dopamine / Sodium:neurotransmitter symporter family signature 2. / Sodium:neurotransmitter symporter family signature 1. / Sodium:neurotransmitter symporter / Sodium:neurotransmitter symporter superfamily / Sodium:neurotransmitter symporter family / Sodium:neurotransmitter symporter family profile.
Similarity search - Domain/homology
CHOLESTEROL / COCAINE / CHOLESTEROL HEMISUCCINATE / Sodium-dependent dopamine transporter
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.66 Å
AuthorsNielsen, J.C. / Salomon, K. / Kalenderoglou, I.E. / Bargmeyer, S. / Pape, T. / Shahsavar, A. / Loland, C.J.
Funding support Denmark, European Union, 6items
OrganizationGrant numberCountry
LundbeckfondenR344-2020-1020 Denmark
The Carlsberg FoundationCF20-0345 Denmark
Independent Research Fund Denmark - Technology and Production Sciences3101-00381B Denmark
H2020 Marie Curie Actions of the European Commission860954European Union
Maersk FoundationL-2022-00324 Denmark
Maersk FoundationL-2021-00122 Denmark
CitationJournal: To Be Published
Title: The molecular basis for cocaine binding to the human dopamine transporter
Authors: Nielsen, J.C. / Salomon, K. / Kalenderoglou, I.E. / Bargmeyer, S. / Pape, T. / Shahsavar, A. / Loland, C.J.
History
DepositionMar 14, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 3, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Sodium-dependent dopamine transporter
hetero molecules


Theoretical massNumber of molelcules
Total (without water)74,2188
Polymers72,2091
Non-polymers2,0087
Water724
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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Protein , 1 types, 1 molecules B

#1: Protein Sodium-dependent dopamine transporter / DA transporter / DAT / Solute carrier family 6 member 3


Mass: 72209.281 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: triple tandem GGGS linker and Twin-Strep tag / Source: (gene. exp.) Homo sapiens (human) / Gene: SLC6A3, DAT1 / Production host: Homo sapiens (human) / References: UniProt: Q01959

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Non-polymers , 6 types, 11 molecules

#2: Chemical ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C27H46O
#3: Chemical ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C31H50O4
#4: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na
#5: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#6: Chemical ChemComp-COC / COCAINE


Mass: 303.353 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C17H21NO4 / Feature type: SUBJECT OF INVESTIGATION / Comment: alkaloid*YM
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 4 / 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: Human dopamine transporter bound to cocaine / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.72 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Calibrated magnification: 170700 X / Nominal defocus max: 1800 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3.27 sec. / Electron dose: 60 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 25808
EM imaging opticsEnergyfilter name: TFS Selectris / Energyfilter slit width: 5 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2EPU3.6image acquisition
4cryoSPARCCTF correction
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.66 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1990718 / Symmetry type: POINT

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