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- PDB-8gnk: CryoEM structure of cytosol-facing, substrate-bound ratGAT1 -

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Basic information

Entry
Database: PDB / ID: 8gnk
TitleCryoEM structure of cytosol-facing, substrate-bound ratGAT1
Components
  • Fragment antigen binding light chain
  • Sodium- and chloride-dependent GABA transporter 1
  • fragment antigen binding 9D5 heavy chain
KeywordsMEMBRANE PROTEIN / Neurotransmitter sodium symporter / GABA transporter / solute carrier 6 / secondary active transport
Function / homology
Function and homology information


Na+/Cl- dependent neurotransmitter transporters / Reuptake of GABA / neurotransmitter reuptake / 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 / amino acid:sodium symporter activity / negative regulation of synaptic transmission, GABAergic ...Na+/Cl- dependent neurotransmitter transporters / Reuptake of GABA / neurotransmitter reuptake / 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 / amino acid:sodium symporter activity / negative regulation of synaptic transmission, GABAergic / positive regulation of gamma-aminobutyric acid secretion / response to sucrose / response to purine-containing compound / sodium ion import across plasma membrane / associative learning / sodium ion transmembrane transport / : / GABA-ergic synapse / chloride transmembrane transport / response to organonitrogen compound / : / response to cocaine / learning / response to lead ion / synapse organization / response to organic cyclic compound / response to toxic substance / memory / response to calcium ion / presynapse / response to estradiol / presynaptic membrane / postsynaptic membrane / neuron projection / axon / neuronal cell body / cell surface / 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
GAMMA-AMINO-BUTANOIC ACID / CHOLESTEROL / PHOSPHATIDYLETHANOLAMINE / Sodium- and chloride-dependent GABA transporter 1
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsNayak, S.R. / Joseph, D. / Penmatsa, A.
Funding support India, 1items
OrganizationGrant numberCountry
Department of Biotechnology (DBT, India)IA/I/15/2/502063 India
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: Cryo-EM structure of GABA transporter 1 reveals substrate recognition and transport mechanism.
Authors: Smruti Ranjan Nayak / Deepthi Joseph / Georg Höfner / Archishman Dakua / Arunabh Athreya / Klaus T Wanner / Baruch I Kanner / Aravind Penmatsa /
Abstract: The inhibitory neurotransmitter γ-aminobutyric acid (GABA) is cleared from the synaptic cleft by the sodium- and chloride-coupled GABA transporter GAT1. Inhibition of GAT1 prolongs the GABAergic ...The inhibitory neurotransmitter γ-aminobutyric acid (GABA) is cleared from the synaptic cleft by the sodium- and chloride-coupled GABA transporter GAT1. Inhibition of GAT1 prolongs the GABAergic signaling at the synapse and is a strategy to treat certain forms of epilepsy. In this study, we present the cryo-electron microscopy structure of Rattus norvegicus GABA transporter 1 (rGAT1) at a resolution of 3.1 Å. The structure elucidation was facilitated by epitope transfer of a fragment-antigen binding (Fab) interaction site from the Drosophila dopamine transporter (dDAT) to rGAT1. The structure reveals rGAT1 in a cytosol-facing conformation, with a linear density in the primary binding site that accommodates a molecule of GABA, a displaced ion density proximal to Na site 1 and a bound chloride ion. A unique insertion in TM10 aids the formation of a compact, closed extracellular gate. Besides yielding mechanistic insights into ion and substrate recognition, our study will enable the rational design of specific antiepileptics.
History
DepositionAug 24, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0May 31, 2023Provider: repository / Type: Initial release
Revision 1.1Jul 5, 2023Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Jul 19, 2023Group: Database references / Category: citation / citation_author / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.3Aug 2, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 2.0Nov 15, 2023Group: Atomic model / Data collection / Category: atom_site / chem_comp_atom / chem_comp_bond / Item: _atom_site.auth_atom_id / _atom_site.label_atom_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Sodium- and chloride-dependent GABA transporter 1
L: Fragment antigen binding light chain
H: fragment antigen binding 9D5 heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)113,78515
Polymers110,1273
Non-polymers3,65812
Water1086
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Antibody , 2 types, 2 molecules LH

#2: Antibody Fragment antigen binding 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)
#3: Antibody fragment antigen binding 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) / Production host: Spodoptera frugiperda (fall armyworm)

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Protein / Sugars , 2 types, 3 molecules A

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


Mass: 63200.676 Da / Num. of mol.: 1
Mutation: F312Y, Y481S, D482E, N483D, Q485R, E486D, V488I, S490F, R491P
Source method: isolated from a genetically manipulated source
Details: ratGAT1 construct / Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Gabt1, Gat-1, Gat1 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: P23978
#4: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 6 types, 16 molecules

#5: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#6: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na
#7: Chemical ChemComp-ABU / GAMMA-AMINO-BUTANOIC ACID / GAMMA(AMINO)-BUTYRIC ACID


Mass: 103.120 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H9NO2 / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical
ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C27H46O
#9: Chemical ChemComp-PTY / PHOSPHATIDYLETHANOLAMINE


Mass: 734.039 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C40H80NO8P / Comment: phospholipid*YM
#10: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Complex of epitope engineered ratGAT1-Fab9d5COMPLEXrGAT1#1-#30RECOMBINANT
2ratGAT1COMPLEX#11RECOMBINANT
3fragment antigen binding 9D5 heavy/light chainCOMPLEX#2-#31RECOMBINANT
Molecular weightValue: 111 kDa/nm / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDCellular location
21Rattus norvegicus (Norway rat)10116membrane
32Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCellPlasmid
21Homo sapiens (human)9606HEK293pEGBacmam
32Spodoptera frugiperda (fall armyworm)7108
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris1
2300 mMsodium chlorideNaCl1
32 %Glycerol1
41 mMDDM1
50.1 mMCHS1
SpecimenConc.: 3.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The sample was a one to one complex of ratGAT1 with an antibody fragment and was homogenous.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Calibrated magnification: 105000 X / Nominal defocus max: 4000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 100 K / Temperature (min): 100 K
Image recordingAverage exposure time: 2.7 sec. / Electron dose: 50.09 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 18152
Details: Images were collected in movie mode at 50 frames per image
EM imaging opticsEnergyfilter name: GIF Bioquantum / Chromatic aberration corrector: No applicable / Energyfilter slit width: 20 eV / Phase plate: OTHER / Spherical aberration corrector: Not applicable

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Processing

SoftwareName: PHENIX / Version: 1.20rc4_4425: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1cryoSPARC3particle selectionblobpicker
2EPU2.14image acquisition
4MotionCorr22CTF correctionPatch motion correction
7Coot0.9model fitting
9PHENIXmodel refinementphenix.refine
12cryoSPARC3classificationAbinitio reconstruction
13cryoSPARC33D reconstructionNon-uniform refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 5838634
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 872611 / Num. of class averages: 3 / Symmetry type: POINT
Atomic model buildingB value: 165 / Protocol: OTHER / Space: REAL / Target criteria: Correlation coefficient
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0036346
ELECTRON MICROSCOPYf_angle_d0.5068649
ELECTRON MICROSCOPYf_dihedral_angle_d7.999924
ELECTRON MICROSCOPYf_chiral_restr0.039964
ELECTRON MICROSCOPYf_plane_restr0.0041021

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