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- EMDB-25170: Human wildtype GABA reuptake transporter 1 in complex with tiagab... -

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Basic information

Entry
Database: EMDB / ID: EMD-25170
TitleHuman wildtype GABA reuptake transporter 1 in complex with tiagabine, inward-open conformation
Map data
Sample
  • Complex: GAT-1 in complex with tiagabine
    • Protein or peptide: Sodium- and chloride-dependent GABA transporter 1
  • Ligand: Tiagabine
Function / homology
Function and homology information


gamma-aminobutyric acid reuptake / Reuptake of GABA / gamma-aminobutyric acid:sodium:chloride symporter activity / sodium:chloride symporter activity / gamma-aminobutyric acid transmembrane transporter activity / gamma-aminobutyric acid import / inorganic anion import across plasma membrane / negative regulation of synaptic transmission, GABAergic / positive regulation of gamma-aminobutyric acid secretion / response to sucrose ...gamma-aminobutyric acid reuptake / Reuptake of GABA / gamma-aminobutyric acid:sodium:chloride symporter activity / sodium:chloride symporter activity / gamma-aminobutyric acid transmembrane transporter activity / gamma-aminobutyric acid import / inorganic anion import across plasma membrane / negative regulation of synaptic transmission, GABAergic / positive regulation of gamma-aminobutyric acid secretion / response to sucrose / response to purine-containing compound / Na+/Cl- dependent neurotransmitter transporters / sodium ion import across plasma membrane / amino acid transport / associative learning / transport across blood-brain barrier / sodium ion transmembrane transport / : / GABA-ergic synapse / chloride transmembrane transport / response to cocaine / response to lead ion / synapse organization / response to toxic substance / memory / response to calcium ion / response to estradiol / presynaptic membrane / chemical synaptic transmission / postsynaptic membrane / axon / neuronal cell body / cell surface / membrane / identical protein binding / metal ion binding / plasma membrane
Similarity search - Function
Sodium:neurotransmitter symporter, GABA, GAT-1 / 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- and chloride-dependent GABA transporter 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.82 Å
AuthorsGati C / Motiwala Z / Aduri NG / Shaye H / Han GW / Cherezov V
Funding support1 items
OrganizationGrant numberCountry
Other private
CitationJournal: Nature / Year: 2022
Title: Structural basis of GABA reuptake inhibition.
Authors: Zenia Motiwala / Nanda Gowtham Aduri / Hamidreza Shaye / Gye Won Han / Jordy Homing Lam / Vsevolod Katritch / Vadim Cherezov / Cornelius Gati /
Abstract: γ-Aminobutyric acid (GABA) transporter 1 (GAT1) regulates neuronal excitation of the central nervous system by clearing the synaptic cleft of the inhibitory neurotransmitter GABA upon its release ...γ-Aminobutyric acid (GABA) transporter 1 (GAT1) regulates neuronal excitation of the central nervous system by clearing the synaptic cleft of the inhibitory neurotransmitter GABA upon its release from synaptic vesicles. Elevating the levels of GABA in the synaptic cleft, by inhibiting GABA reuptake transporters, is an established strategy to treat neurological disorders, such as epilepsy. Here we determined the cryo-electron microscopy structure of full-length, wild-type human GAT1 in complex with its clinically used inhibitor tiagabine, with an ordered part of only 60 kDa. Our structure reveals that tiagabine locks GAT1 in the inward-open conformation, by blocking the intracellular gate of the GABA release pathway, and thus suppresses neurotransmitter uptake. Our results provide insights into the mixed-type inhibition of GAT1 by tiagabine, which is an important anticonvulsant medication. Its pharmacodynamic profile, confirmed by our experimental data, suggests initial binding of tiagabine to the substrate-binding site in the outward-open conformation, whereas our structure presents the drug stalling the transporter in the inward-open conformation, consistent with a two-step mechanism of inhibition. The presented structure of GAT1 gives crucial insights into the biology and pharmacology of this important neurotransmitter transporter and provides blueprints for the rational design of neuromodulators, as well as moving the boundaries of what is considered possible in single-particle cryo-electron microscopy of challenging membrane proteins.
History
DepositionOct 19, 2021-
Header (metadata) releaseJun 8, 2022-
Map releaseJun 8, 2022-
UpdateJul 6, 2022-
Current statusJul 6, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25170.png

Downloads & links

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Map

FileDownload / File: emd_25170.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.813 Å
Density
Contour LevelBy AUTHOR: 0.11
Minimum - Maximum-0.41715845 - 0.67828023
Average (Standard dev.)-0.0003649103 (±0.018955061)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 208.128 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : GAT-1 in complex with tiagabine

EntireName: GAT-1 in complex with tiagabine
Components
  • Complex: GAT-1 in complex with tiagabine
    • Protein or peptide: Sodium- and chloride-dependent GABA transporter 1
  • Ligand: Tiagabine

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Supramolecule #1: GAT-1 in complex with tiagabine

SupramoleculeName: GAT-1 in complex with tiagabine / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
Molecular weightExperimental: 67 kDa/nm

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Macromolecule #1: Sodium- and chloride-dependent GABA transporter 1

MacromoleculeName: Sodium- and chloride-dependent GABA transporter 1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 64.886793 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MATNGSKVAD GQISTEVSEA PVANDKPKTL VVKVQKKAAD LPDRDTWKGR FDFLMSCVGY AIGLGNVWRF PYLCGKNGGG AFLIPYFLT LIFAGVPLFL LECSLGQYTS IGGLGVWKLA PMFKGVGLAA AVLSFWLNIY YIVIISWAIY YLYNSFTTTL P WKQCDNPW ...String:
MATNGSKVAD GQISTEVSEA PVANDKPKTL VVKVQKKAAD LPDRDTWKGR FDFLMSCVGY AIGLGNVWRF PYLCGKNGGG AFLIPYFLT LIFAGVPLFL LECSLGQYTS IGGLGVWKLA PMFKGVGLAA AVLSFWLNIY YIVIISWAIY YLYNSFTTTL P WKQCDNPW NTDRCFSNYS MVNTTNMTSA VVEFWERNMH QMTDGLDKPG QIRWPLAITL AIAWILVYFC IWKGVGWTGK VV YFSATYP YIMLIILFFR GVTLPGAKEG ILFYITPNFR KLSDSEVWLD AATQIFFSYG LGLGSLIALG SYNSFHNNVY RDS IIVCCI NSCTSMFAGF VIFSIVGFMA HVTKRSIADV AASGPGLAFL AYPEAVTQLP ISPLWAILFF SMLLMLGIDS QFCT VEGFI TALVDEYPRL LRNRRELFIA AVCIISYLIG LSNITQGGIY VFKLFDYYSA SGMSLLFLVF FECVSISWFY GVNRF YDNI QEMVGSRPCI WWKLCWSFFT PIIVAGVFIF SAVQMTPLTM GNYVFPKWGQ GVGWLMALSS MVLIPGYMAY MFLTLK GSL KQRIQVMVQP SEDIV

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Macromolecule #2: Tiagabine

MacromoleculeName: Tiagabine / type: ligand / ID: 2 / Number of copies: 1 / Formula: TGI
Molecular weightTheoretical: 375.548 Da
Chemical component information

ChemComp-TGI:
Tiagabine / medication*YM

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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

Concentration18 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMHEPESHEPES
150.0 mMNaClsodium chloride
0.001 %LMNGdetergent
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 4000.0 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 39572 / 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: 105000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 25.0 µm / Nominal defocus min: 10.0 µm
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

CTF correctionSoftware - Name: cryoSPARC (ver. v3.2.0+210831)
Startup modelType of model: INSILICO MODEL / In silico model: Ab initio
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.82 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v3.2.0+210831) / Number images used: 135297
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. v3.2.0+210831)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. v3.2.0+210831)
Final 3D classificationNumber classes: 2

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