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- PDB-9cp5: Cryo-EM structure of human GAT3, apo state, inward-open conformation -

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Basic information

Entry
Database: PDB / ID: 9cp5
TitleCryo-EM structure of human GAT3, apo state, inward-open conformation
Components
  • 9D5 heavy chain
  • 9D5 light chain
  • Sodium- and chloride-dependent GABA transporter 3
KeywordsMEMBRANE PROTEIN / GABA transporter 3 / GAT3 / SLC6 / neurotransmitter transporter / NSS / cryo-EM / single particle / astrocyte
Function / homology
Function and homology information


gamma-aminobutyric acid reuptake / Reuptake of GABA / monocarboxylic acid transmembrane transporter activity / monocarboxylic acid transport / Creatine metabolism / taurine:sodium symporter activity / gamma-aminobutyric acid:sodium:chloride symporter activity / SLC-mediated transport of neurotransmitters / amino acid binding / amino acid transport ...gamma-aminobutyric acid reuptake / Reuptake of GABA / monocarboxylic acid transmembrane transporter activity / monocarboxylic acid transport / Creatine metabolism / taurine:sodium symporter activity / gamma-aminobutyric acid:sodium:chloride symporter activity / SLC-mediated transport of neurotransmitters / amino acid binding / amino acid transport / sodium ion transmembrane transport / cell projection / GABA-ergic synapse / presynaptic membrane / postsynaptic membrane / response to xenobiotic stimulus / membrane / plasma membrane
Similarity search - Function
Sodium:neurotransmitter symporter, GABA, GAT-3 / 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 3
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.51 Å
AuthorsYadav, R. / Han, G.W. / Gati, C.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Commun / Year: 2025
Title: Molecular basis of human GABA transporter 3 inhibition.
Authors: Ravi Yadav / Gye Won Han / Cornelius Gati /
Abstract: γ-Aminobutyric acid (GABA) transporters (GATs) are sodium- and chloride-dependent transporters that mediate the reuptake of the inhibitory neurotransmitter GABA after its release from synaptic ...γ-Aminobutyric acid (GABA) transporters (GATs) are sodium- and chloride-dependent transporters that mediate the reuptake of the inhibitory neurotransmitter GABA after its release from synaptic vesicles. GAT3 transports GABA from the synaptic cleft into astrocytes and modulates synaptic signaling. GAT3 has been implicated in various neurological disorders and neurodegenerative diseases, rendering it a therapeutically important drug target. To understand the mechanism of transport and inhibition, here we determine cryo-electron microscopy structures of human GAT3 in its apo form and in complex with the selective inhibitor SNAP-5114. Unexpectedly, we have discovered that SNAP-5114 acts as a noncompetitive inhibitor at GAT3. SNAP-5114 binds at the orthosteric substrate binding pocket of GAT3 in its inward-open conformation, in agreement with its noncompetitive inhibition of GABA transport. In the apo state, GAT3 also adopts an inward-open conformation with the orthosteric substrate binding pocket exposed to cytoplasm, while an extensive network of interactions closes the extracellular gate. The structures, complemented with mutagenesis and radioligand uptake assays, show that the increased orthosteric substrate binding pocket volume and bulky moieties of SNAP-5114, drive the selective inhibition of GAT3 over GAT1. Our structural and functional studies reveal the mechanism of selective inhibition of GAT3 and provide a framework for GAT3-targeted rational drug design.
History
DepositionJul 17, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 7, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Sodium- and chloride-dependent GABA transporter 3
H: 9D5 heavy chain
L: 9D5 light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)121,1034
Polymers121,0673
Non-polymers351
Water00
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

#1: Protein Sodium- and chloride-dependent GABA transporter 3 / GAT-3 / Solute carrier family 6 member 11


Mass: 71518.500 Da / Num. of mol.: 1 / Mutation: N327H, Y497S, D498E, N499D, E501R, Y506F, R507P
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC6A11, GABT3, GAT3 / Plasmid: BacMam / Cell line (production host): HEK293S GnTI- / Production host: Homo sapiens (human) / References: UniProt: P48066
#2: Antibody 9D5 heavy chain


Mass: 26242.297 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)
#3: Antibody 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) / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm)
#4: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationY

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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 GABA transporter 3 in complex with 9D5 / Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.111 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293S GnTI- / Plasmid: BacMam
Buffer solutionpH: 7.5
SpecimenConc.: 3.274 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil 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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 3400 nm / Nominal defocus min: 800 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: 1.8 sec. / Electron dose: 52.5 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 9115
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARCv4.5.3particle selection
2EPUimage acquisition
4cryoSPARCv4.5.3CTF correction
7UCSF ChimeraXmodel fitting
9cryoSPARCv4.5.3initial Euler assignment
10cryoSPARCv4.5.3final Euler assignment
11cryoSPARCv4.5.3classification
12cryoSPARCv4.5.33D reconstruction
13PHENIX1.21.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 6319538
3D reconstructionResolution: 3.51 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 158075 / Symmetry type: POINT
Atomic model buildingDetails: D_1000286001 structure / Source name: Other / Type: other
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0037659
ELECTRON MICROSCOPYf_angle_d0.71110444
ELECTRON MICROSCOPYf_dihedral_angle_d4.3681039
ELECTRON MICROSCOPYf_chiral_restr0.0481178
ELECTRON MICROSCOPYf_plane_restr0.0051292

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