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- EMDB-19845: Outward-open structure of human dopamine transporter bound to cocaine -

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Basic information

Entry
Database: EMDB / ID: EMD-19845
TitleOutward-open structure of human dopamine transporter bound to cocaine
Map dataMAP SHARP
Sample
  • Complex: Human dopamine transporter bound to cocaine
    • Protein or peptide: Sodium-dependent dopamine transporter
  • Ligand: CHOLESTEROL
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: SODIUM ION
  • Ligand: CHLORIDE ION
  • Ligand: COCAINE
  • Ligand: water
Keywordsneurotransmitter/sodium symporters / monoamine transporter / dopamine transporter / SLC6 / MEMBRANE PROTEIN
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 / dopamine uptake / adenohypophysis development / hyaloid vascular plexus regression / dopamine binding / norepinephrine:sodium symporter activity / dopamine: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 / dopamine uptake / adenohypophysis development / hyaloid vascular plexus regression / dopamine binding / norepinephrine:sodium symporter activity / dopamine:sodium symporter activity / neurotransmitter transmembrane transporter activity / regulation of dopamine metabolic process / dopamine transport / flotillin complex / dopamine catabolic process / dopaminergic synapse / monoamine transmembrane transporter activity / monoamine transport / positive regulation of multicellular organism growth / SLC-mediated transport of neurotransmitters / heterocyclic compound binding / response to iron ion / dopamine biosynthetic process / neurotransmitter transport / amino acid transport / dopamine uptake involved in synaptic transmission / response to cAMP / prepulse inhibition / lactation / axon terminus / sodium ion transmembrane transport / protein phosphatase 2A binding / response to nicotine / response to cocaine / locomotory behavior / cognition / sensory perception of smell / presynaptic membrane / protease binding / response to ethanol / postsynaptic membrane / neuron projection / membrane raft / response to xenobiotic stimulus / signaling receptor binding / axon / neuronal cell body / protein-containing complex binding / cell surface / metal ion binding / membrane / 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
Sodium-dependent dopamine transporter
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.66 Å
AuthorsNielsen JC / Salomon K / Kalenderoglou IE / Bargmeyer S / Pape T / Shahsavar A / Loland CJ
Funding support Denmark, European Union, 6 items
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: Nature / Year: 2024
Title: Structure of the human dopamine transporter in complex with cocaine.
Authors: Jeppe C Nielsen / Kristine Salomon / Iris E Kalenderoglou / Sarah Bargmeyer / Tillmann Pape / Azadeh Shahsavar / Claus J Loland /
Abstract: The dopamine transporter (DAT) is crucial for regulating dopamine signalling and is the prime mediator for the rewarding and addictive effects of cocaine. As part of the neurotransmitter sodium ...The dopamine transporter (DAT) is crucial for regulating dopamine signalling and is the prime mediator for the rewarding and addictive effects of cocaine. As part of the neurotransmitter sodium symporter family, DAT uses the Na gradient across cell membranes to transport dopamine against its chemical gradient. The transport mechanism involves both intra- and extracellular gates that control substrate access to a central site. However, the molecular intricacies of this process and the inhibitory mechanism of cocaine have remained unclear. Here, we present the molecular structure of human DAT in complex with cocaine at a resolution of 2.66 Å. Our findings reveal that DAT adopts the expected LeuT-fold, posing in an outward-open conformation with cocaine bound at the central (S1) site. Notably, while an Na occupies the second Na site (Na2), the Na1 site seems to be vacant, with the side chain of Asn82 occupying the presumed Na space. This structural insight elucidates the mechanism for the cocaine inhibition of human DAT and deepens our understanding of neurotransmitter transport. By shedding light on the molecular underpinnings of how cocaine acts, our study lays a foundation for the development of targeted medications to combat addiction.
History
DepositionMar 14, 2024-
Header (metadata) releaseJul 3, 2024-
Map releaseJul 3, 2024-
UpdateNov 13, 2024-
Current statusNov 13, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19845.png

Downloads & links

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Map

FileDownload / File: emd_19845.map.gz / Format: CCP4 / Size: 166.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMAP SHARP
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.73 Å/pix.
x 352 pix.
= 255.2 Å		0.73 Å/pix.
x 352 pix.
= 255.2 Å		0.73 Å/pix.
x 352 pix.
= 255.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.725 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.216
Minimum - Maximum-1.0976113 - 1.6164905
Average (Standard dev.)-0.0009672735 (±0.033638816)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions352352352
Spacing352352352
CellA=B=C: 255.20001 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_19845_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: EM MAP

Fileemd_19845_additional_1.map
AnnotationEM MAP
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: HALF MAP B

Fileemd_19845_half_map_1.map
AnnotationHALF MAP B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: HALF MAP A

Fileemd_19845_half_map_2.map
AnnotationHALF MAP A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Human dopamine transporter bound to cocaine

EntireName: Human dopamine transporter bound to cocaine
Components
  • Complex: Human dopamine transporter bound to cocaine
    • Protein or peptide: Sodium-dependent dopamine transporter
  • Ligand: CHOLESTEROL
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: SODIUM ION
  • Ligand: CHLORIDE ION
  • Ligand: COCAINE
  • Ligand: water

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Supramolecule #1: Human dopamine transporter bound to cocaine

SupramoleculeName: Human dopamine transporter bound to cocaine / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 720 KDa

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Macromolecule #1: Sodium-dependent dopamine transporter

MacromoleculeName: Sodium-dependent dopamine transporter / type: protein_or_peptide / ID: 1 / Details: triple tandem GGGS linker and Twin-Strep tag / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 72.209281 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MSKSKCSVGL MSSVVAPAKE PNAVGPKEVE LILVKEQNGV QLTSSTLTNP RQSPVEAQDR ETWGKKIDFL LSVIGFAVDL ANVWRFPYL CYKNGGGAFL VPYLLFMVIA GMPLFYMELA LGQFNREGAA GVWKICPILK GVGFTVILIS LYVGFFYNVI I AWALHYLF ...String:
MSKSKCSVGL MSSVVAPAKE PNAVGPKEVE LILVKEQNGV QLTSSTLTNP RQSPVEAQDR ETWGKKIDFL LSVIGFAVDL ANVWRFPYL CYKNGGGAFL VPYLLFMVIA GMPLFYMELA LGQFNREGAA GVWKICPILK GVGFTVILIS LYVGFFYNVI I AWALHYLF SSFTTELPWI HCNNSWNSPN CSDAHPGDSS GDSSGLNDTF GTTPAAEYFE RGVLHLHQSH GIDDLGPPRW QL TACLVLV IVLLYFSLWK GVKTSGKVVW ITATMPYVVL TALLLRGVTL PGAIDGIRAY LSVDFYRLCE ASVWIDAATQ VCF SLGVGF GVLIAFSSYN KFTNNCYRDA IVTTSINSLT SFSSGFVVFS FLGYMAQKHS VPIGDVAKDG PGLIFIIYPE AIAT LPLSS AWAVVFFIML LTLGIDSAMG GMESVITGLI DEFQLLHRHR ELFTLFIVLA TFLLSLFCVT NGGIYVFTLL DHFAA GTSI LFGVLIEAIG VAWFYGVGQF SDDIQQMTGQ RPSLYWRLCW KLVSPCFLLF VVVVSIVTFR PPHYGAYIFP DWANAL GWV IATSSMAMVP IYAAYKFCSL PGSFREKLAY AIAPEKDREL VDRGEVRQFT LRHWLKVGGG SGGGSGGSAW SHPQFEK GG GSGGGSGGSA WSHPQFEK

UniProtKB: Sodium-dependent dopamine transporter

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Macromolecule #2: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 2 / Number of copies: 3 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL

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Macromolecule #3: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 3 / Number of copies: 1 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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Macromolecule #4: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 4 / Number of copies: 1
Molecular weightTheoretical: 22.99 Da

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Macromolecule #5: CHLORIDE ION

MacromoleculeName: CHLORIDE ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: CL
Molecular weightTheoretical: 35.453 Da

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Macromolecule #6: COCAINE

MacromoleculeName: COCAINE / type: ligand / ID: 6 / Number of copies: 1 / Formula: COC
Molecular weightTheoretical: 303.353 Da
Chemical component information

ChemComp-COC:
COCAINE

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Macromolecule #7: water

MacromoleculeName: water / type: ligand / ID: 7 / Number of copies: 4 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Pressure: 0.0001 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris / Energy filter - Slit width: 5 eV
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 25808 / Average exposure time: 3.27 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 170700 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 165000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE / Details: Alphafold model
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.66 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 1990718
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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