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

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Basic information

Entry
Database: PDB / ID: 7wgd
TitleX-ray structure of thermostabilized Drosophila dopamine transporter with GABA transporter1-like substitutions in the binding site, in substrate-free form.
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 / Na+/Cl- dependent neurotransmitter transporters / dopamine:sodium symporter activity / norepinephrine transport / circadian sleep/wake cycle / response to odorant / cocaine binding / regulation of presynaptic cytosolic calcium ion concentration / dopamine transport / sleep ...Dopamine clearance from the synaptic cleft / Na+/Cl- dependent neurotransmitter transporters / dopamine:sodium symporter activity / norepinephrine transport / circadian sleep/wake cycle / response to odorant / cocaine binding / regulation of presynaptic cytosolic calcium ion concentration / dopamine transport / sleep / dopamine uptake involved in synaptic transmission / neuronal cell body membrane / sodium ion transmembrane transport / adult locomotory behavior / presynaptic membrane / neuron projection / 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.
Similarity search - Domain/homology
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: 3.2 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

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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)108,0258
Polymers107,0713
Non-polymers9555
Water34219
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, monomer complexed with antibody fragment
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)96.388, 140.709, 168.021
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, V120L, D121N, S422Q, G425T, S426V, A117S, F325L, V327S, E384S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly) / Gene: DAT, fmn, CG8380 / Plasmid: pEG Bacmam / 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: pFastbac 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) / Plasmid: pFastbac dual / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm)

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Non-polymers , 5 types, 24 molecules

#4: Chemical ChemComp-NA / SODIUM ION


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


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


Mass: 486.726 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C31H50O4
#7: Chemical ChemComp-CLR / CHOLESTEROL / Cholesterol


Mass: 386.654 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H46O
#8: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 19 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN

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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: 5.33 Å3/Da / Density % sol: 78 % / Description: long rods
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 7.2 / Details: 0.1M MOPS pH 7.2, 36% PEG 600 / PH range: 7.0-7.5 / Temp details: cold room crystallization (4-6C)

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Data collection

DiffractionMean temperature: 100 K / Ambient temp details: cryo stream at 100K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.9792 Å
DetectorType: DECTRIS EIGER 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: 3.2→48.43 Å / Num. obs: 38565 / % possible obs: 99.8 % / Redundancy: 12.9 % / Biso Wilson estimate: 95.67 Å2 / CC1/2: 0.914 / Rmerge(I) obs: 0.717 / Rpim(I) all: 0.219 / Rrim(I) all: 0.753 / Net I/σ(I): 9.1 / Num. measured all: 497543 / Scaling rejects: 14930
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
3.2-3.3413.65.5476271445990.4281.5475.7631.298.8
11.07-48.4312.10.0531248610350.9990.0160.05633.198.3

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Processing

Software
NameVersionClassification
PHENIXv1.20rc4-4425refinement
XDSdata reduction
Aimless0.7.3data scaling
PDB_EXTRACT3.27data extraction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4XNU

4xnu


Resolution: 3.2→48.43 Å / SU ML: 0.45 / Cross valid method: THROUGHOUT / σ(F): 1.33 / Phase error: 30.76 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2864 1910 4.99 %
Rwork0.2436 36376 -
obs0.2456 38286 99.24 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 177.35 Å2 / Biso mean: 85.9383 Å2 / Biso min: 51.84 Å2
Refinement stepCycle: final / Resolution: 3.2→48.43 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7481 0 66 19 7566
Biso mean--93.44 77.43 -
Num. residues----969
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 14

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
3.2-3.280.33031270.33072550267798
3.28-3.360.34791160.313125942710100
3.36-3.460.29381310.289225822713100
3.46-3.570.34921490.290525652714100
3.57-3.70.31061440.26425652709100
3.7-3.850.26041290.240225942723100
3.85-4.030.27861380.22925772715100
4.03-4.240.26811350.22462552268799
4.24-4.50.26851460.22062544269098
4.5-4.850.2481270.20962583271098
4.85-5.340.29081390.21952602274199
5.34-6.110.25761490.23626082757100
6.11-7.690.2651330.245126742807100
7.69-48.430.32051470.2552786293399

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