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- EMDB-45799: Cryo-EM structure of human GAT3 in complex with SNAP-5114, inward... -

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Basic information

Entry
Database: EMDB / ID: EMD-45799
TitleCryo-EM structure of human GAT3 in complex with SNAP-5114, inward-open conformation
Map data
Sample
  • Complex: Human GABA transporter 3 in complex with 9D5 and SNAP-5114
    • Protein or peptide: Sodium- and chloride-dependent GABA transporter 3
    • Protein or peptide: 9D5 heavy chain
    • Protein or peptide: 9D5 light chain
  • Ligand: (3S)-1-{2-[tris(4-methoxyphenyl)methoxy]ethyl}piperidine-3-carboxylic acid
  • Ligand: CHLORIDE ION
KeywordsGABA transporter 3 / GAT3 / SNAP-5114 / SLC6 / neurotransmitter transporter / NSS / cryo-EM / single particle / astrocyte / MEMBRANE PROTEIN
Function / homology
Function and homology information


gamma-aminobutyric acid reuptake / Reuptake of GABA / taurine:sodium symporter activity / monocarboxylic acid transmembrane transporter activity / monocarboxylic acid transport / gamma-aminobutyric acid:sodium:chloride symporter activity / Creatine metabolism / Na+/Cl- dependent neurotransmitter transporters / amino acid binding / amino acid transport ...gamma-aminobutyric acid reuptake / Reuptake of GABA / taurine:sodium symporter activity / monocarboxylic acid transmembrane transporter activity / monocarboxylic acid transport / gamma-aminobutyric acid:sodium:chloride symporter activity / Creatine metabolism / Na+/Cl- dependent neurotransmitter transporters / amino acid binding / amino acid transport / sodium ion transmembrane transport / GABA-ergic synapse / cell projection / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.42 Å
AuthorsYadav R / Han GW / Gati C
Funding support1 items
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, 2024-
Header (metadata) releaseMay 7, 2025-
Map releaseMay 7, 2025-
UpdateMay 7, 2025-
Current statusMay 7, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45799.png

Downloads & links

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Map

FileDownload / File: emd_45799.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 512 pix.
= 331.264 Å		0.65 Å/pix.
x 512 pix.
= 331.264 Å		0.65 Å/pix.
x 512 pix.
= 331.264 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.647 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.275
Minimum - Maximum-1.0914412 - 1.6716107
Average (Standard dev.)-0.0013500017 (±0.029448666)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 331.264 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_45799_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_45799_half_map_2.map
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 GABA transporter 3 in complex with 9D5 and SNAP-5114

EntireName: Human GABA transporter 3 in complex with 9D5 and SNAP-5114
Components
  • Complex: Human GABA transporter 3 in complex with 9D5 and SNAP-5114
    • Protein or peptide: Sodium- and chloride-dependent GABA transporter 3
    • Protein or peptide: 9D5 heavy chain
    • Protein or peptide: 9D5 light chain
  • Ligand: (3S)-1-{2-[tris(4-methoxyphenyl)methoxy]ethyl}piperidine-3-carboxylic acid
  • Ligand: CHLORIDE ION

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Supramolecule #1: Human GABA transporter 3 in complex with 9D5 and SNAP-5114

SupramoleculeName: Human GABA transporter 3 in complex with 9D5 and SNAP-5114
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 111 KDa

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Macromolecule #1: Sodium- and chloride-dependent GABA transporter 3

MacromoleculeName: Sodium- and chloride-dependent GABA transporter 3 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 71.5185 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MTAEKALPLG NGKAAEEARE SEAPGGGCSS GGAAPARHPR VKRDKAVHER GHWNNKVEFV LSVAGEIIGL GNVWRFPYLC YKNGGGAFL IPYVVFFICC GIPVFFLETA LGQFTSEGGI TCWRKVCPLF EGIGYATQVI EAHLNVYYII ILAWAIFYLS N CFTTELPW ...String:
MTAEKALPLG NGKAAEEARE SEAPGGGCSS GGAAPARHPR VKRDKAVHER GHWNNKVEFV LSVAGEIIGL GNVWRFPYLC YKNGGGAFL IPYVVFFICC GIPVFFLETA LGQFTSEGGI TCWRKVCPLF EGIGYATQVI EAHLNVYYII ILAWAIFYLS N CFTTELPW ATCGHEWNTE NCVEFQKLNV SNYSHVSLQN ATSPVMEFWE HRVLAISDGI EHIGNLRWEL ALCLLAAWTI CY FCIWKGT KSTGKVVYVT ATFPYIMLLI LLIRGVTLPG ASEGIKFYLY PDLSRLSDPQ VWVDAGTQIF FSYAICLGCL TAL GSYNNY HNNCYRDCIM LCCLNSGTSF VAGFAIFSVL GFMAYEQGVP IAEVAESGPG LAFIAYPKAV TMMPLSPLWA TLFF MMLIF LGLDSQFVCV ESLVTAVVDM YPKVFRRGYR RELLILALSV ISYFLGLVML TEGGMYIFQL FDSYAASGMC LLFVA IFEC ICIGWVYGSN RFSEDIRDMI GFPPPSLIKW CWMIMTPGIC AGIFIFFLIK YKPLKYNNIY TYPAWGYGIG WLMALS SML CIPLWICITV WKTEGTLPEK LQKLTTPSTD LKMRGKLGVS PRMVTVNDCD AKLKSDGTIA AITEKETHFA AALEVLF Q

UniProtKB: Sodium- and chloride-dependent GABA transporter 3

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Macromolecule #2: 9D5 heavy chain

MacromoleculeName: 9D5 heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 26.242297 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: EVQLVESGGG LVKPGGSLKL SCAASGFTFS SYAMSWVRQS PEKRLEWVAE ISSGGRYIYY SDTVTGRFTI SRDNARNILH LEMSSLRSE DTAMYYCARG EVRQRGFDYW GQGTTLTVSS AKTTAPSVYP LAPVCGDTTG SSVTLGCLVK GYFPEPVTLT W NSGSLSSG ...String:
EVQLVESGGG LVKPGGSLKL SCAASGFTFS SYAMSWVRQS PEKRLEWVAE ISSGGRYIYY SDTVTGRFTI SRDNARNILH LEMSSLRSE DTAMYYCARG EVRQRGFDYW GQGTTLTVSS AKTTAPSVYP LAPVCGDTTG SSVTLGCLVK GYFPEPVTLT W NSGSLSSG VHTFPAVLQS DLYTLSSSVT VTSSTWPSQS ITCNVAHPAS STKVDKKIEP RAAALEVLFQ GPGSHHHHHH HH HH

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Macromolecule #3: 9D5 light chain

MacromoleculeName: 9D5 light chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 23.306586 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: ENVLTQSPAI MSTSPGEKVT MTCRASSSVG SSYLHWYQQK SGASPKLWIY STSNLASGVP ARFSGSGSGT SYSLTISSVE AEDAATYYC QQFSGYPLTF GSGTKLEMKR ADAAPTVSIF PPSSEQLTSG GASVVCFLNN FYPKDINVKW KIDGSERQNG V LNSWTDQD ...String:
ENVLTQSPAI MSTSPGEKVT MTCRASSSVG SSYLHWYQQK SGASPKLWIY STSNLASGVP ARFSGSGSGT SYSLTISSVE AEDAATYYC QQFSGYPLTF GSGTKLEMKR ADAAPTVSIF PPSSEQLTSG GASVVCFLNN FYPKDINVKW KIDGSERQNG V LNSWTDQD SKDSTYSMSS TLTLTKDEYE RHNSYTCEAT HKTSTSPIVK SFNRNE

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Macromolecule #4: (3S)-1-{2-[tris(4-methoxyphenyl)methoxy]ethyl}piperidine-3-carbox...

MacromoleculeName: (3S)-1-{2-[tris(4-methoxyphenyl)methoxy]ethyl}piperidine-3-carboxylic acid
type: ligand / ID: 4 / Number of copies: 1 / Formula: A1AZJ
Molecular weightTheoretical: 505.602 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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration3.646 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS 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: 10524 / Average exposure time: 1.8 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.4 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000
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

Particle selectionNumber selected: 4791432
CTF correctionSoftware - Name: cryoSPARC (ver. v4.3.1) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.42 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v4.3.1) / Number images used: 216211
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. v4.3.1)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. v4.3.1)
Final 3D classificationNumber classes: 4 / Software - Name: cryoSPARC (ver. v4.3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

6-f1

source_name: AlphaFold, initial_model_type: in silico model

4xp5

chain_id: H, source_name: PDB, initial_model_type: experimental model

4xp5

chain_id: L, source_name: PDB, initial_model_type: experimental model
Output model

PDB-9cp4:
Cryo-EM structure of human GAT3 in complex with SNAP-5114, inward-open conformation

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