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- PDB-7wgt: X-ray structure of thermostabilized Drosophila dopamine transport... -

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Basic information

Entry
Database: PDB / ID: 7wgt
TitleX-ray structure of thermostabilized Drosophila dopamine transporter with GABA transporter1-like substitutions in the binding site, in complex with NO711.
Components
  • (Antibody fragment (9D5) ...) x 2
  • Sodium-dependent dopamine transporter
KeywordsMEMBRANE PROTEIN / neurotransmitter transporter / antibody fragment / GABA transporter
Function / homology
Function and homology information


Dopamine clearance from the synaptic cleft / SLC-mediated transport of neurotransmitters / circadian sleep/wake cycle / response to odorant / cocaine binding / norepinephrine transport / dopamine:sodium symporter activity / regulation of presynaptic cytosolic calcium ion concentration / dopamine transport / sleep ...Dopamine clearance from the synaptic cleft / SLC-mediated transport of neurotransmitters / circadian sleep/wake cycle / response to odorant / cocaine binding / norepinephrine transport / dopamine:sodium symporter activity / regulation of presynaptic cytosolic calcium ion concentration / dopamine transport / sleep / neuronal cell body membrane / dopamine uptake involved in synaptic transmission / amino acid transport / sodium ion transmembrane transport / adult locomotory behavior / presynaptic membrane / axon / metal ion binding / plasma membrane
Similarity search - Function
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. / Immunoglobulins / Immunoglobulin-like / Sandwich / Mainly Beta
Similarity search - Domain/homology
Chem-9BC / CHOLESTEROL / CHOLESTEROL HEMISUCCINATE / Sodium-dependent dopamine transporter
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly)
Mus musculus (house mouse)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.75 Å
AuthorsJoseph, D. / Penmatsa, A.
Funding support India, 1items
OrganizationGrant numberCountry
Department of Biotechnology (DBT, India)IA/I/15/2/502063 India
CitationJournal: Embo J. / Year: 2022
Title: Structural insights into GABA transport inhibition using an engineered neurotransmitter transporter.
Authors: Joseph, D. / Nayak, S.R. / Penmatsa, A.
History
DepositionDec 28, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 29, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 20, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Aug 17, 2022Group: Database references / Category: citation / Item: _citation.journal_volume
Revision 1.3Nov 29, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model
Revision 1.4Oct 23, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification
Revision 1.5Mar 12, 2025Group: Database references / Category: pdbx_database_related / Item: _pdbx_database_related.content_type

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Sodium-dependent dopamine transporter
L: Antibody fragment (9D5) light chain
H: Antibody fragment (9D5) heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)108,3769
Polymers107,0713
Non-polymers1,3056
Water50428
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)98.007, 135.025, 162.559
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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Components

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Protein , 1 types, 1 molecules A

#1: Protein Sodium-dependent dopamine transporter / Protein fumin


Mass: 60144.594 Da / Num. of mol.: 1
Mutation: Thermostabilizing (V74A, V275A, V311A, L415A, G538L); GAT1 like (F43Y, D46G, A117S, V120L, D121N, F325L, V327S,S422Q, G425T, S426V, E384S)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly) / Gene: DAT, fmn, CG8380 / Cell line (production host): HEK293S / Production host: Homo sapiens (human) / References: UniProt: Q7K4Y6

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Antibody , 2 types, 2 molecules LH

#2: Antibody Antibody fragment (9D5) light chain


Mass: 23306.586 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Plasmid: pfast bac dual / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm)
#3: Antibody Antibody fragment (9D5) heavy chain


Mass: 23619.430 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm)

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Non-polymers , 6 types, 34 molecules

#4: Chemical ChemComp-9BC / 1-(2-{[(diphenylmethylidene)amino]oxy}ethyl)-1,2,5,6-tetrahydropyridine-3-carboxylic acid / NO-711


Mass: 350.411 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H22N2O3 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H46O
#6: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Na
#7: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#8: Chemical ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C31H50O4
#9: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 28 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 5.02 Å3/Da / Density % sol: 75.51 % / Description: long rods
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 8.2 / Details: 0.1M Tris Bicine pH8.2, 34% PEG 600, 0.5mM NO711 / PH range: 8 - 8.5 / Temp details: cold room 4 to 6 C

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Data collection

DiffractionMean temperature: 100 K / Ambient temp details: cryostream Liq N2 / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.9792 Å
DetectorType: DECTRIS EIGER2 X 16M / Detector: PIXEL / Date: Feb 26, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9792 Å / Relative weight: 1
ReflectionResolution: 2.75→47.42 Å / Num. obs: 56669 / % possible obs: 99.6 % / Redundancy: 5.6 % / Biso Wilson estimate: 93.71 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.128 / Rpim(I) all: 0.058 / Rrim(I) all: 0.141 / Net I/σ(I): 9.8 / Num. measured all: 318635 / Scaling rejects: 356
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. measured allNum. unique obsCC1/2Rpim(I) allRrim(I) allNet I/σ(I) obs% possible all
2.75-2.835.83.3192642845380.3161.4793.6410.698.7
11.66-47.4250.024412483110.0120.02641.196.7

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Processing

Software
NameVersionClassification
PHENIX1.20rc4_4425refinement
XDSdata reduction
Aimless0.7.3data scaling
PDB_EXTRACT3.27data extraction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 7WGD

7wgd


Resolution: 2.75→47.42 Å / SU ML: 0.51 / Cross valid method: THROUGHOUT / σ(F): 1.33 / Phase error: 34.87 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2955 2841 5.03 %
Rwork0.2478 53660 -
obs0.2501 56501 99.44 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 160.74 Å2 / Biso mean: 95.1297 Å2 / Biso min: 57.99 Å2
Refinement stepCycle: final / Resolution: 2.75→47.42 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7514 0 66 28 7608
Biso mean--102.85 92.6 -
Num. residues----970
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 20

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.75-2.80.45331440.40252570271497
2.8-2.850.44231310.39062620275199
2.85-2.90.39941420.361526712813100
2.9-2.960.35771490.340226342783100
2.96-3.030.35741520.334726642816100
3.03-3.10.41181370.353926602797100
3.1-3.170.40861270.348326932820100
3.17-3.260.38341360.32682656279299
3.26-3.360.3261110.30072688279999
3.36-3.460.31111450.273826642809100
3.46-3.590.30731460.2726732819100
3.59-3.730.30821460.27226722818100
3.73-3.90.27371370.251226922829100
3.9-4.110.30131460.243526972843100
4.11-4.360.26511490.21262665281499
4.36-4.70.27311580.202826902848100
4.7-5.170.22251380.19442710284899
5.17-5.920.27551510.219827102861100
5.92-7.450.26531660.245127542920100
7.45-47.420.31211300.23992877300798

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