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- PDB-8g4n: Native GABA-A receptor from the mouse brain, alpha1-beta2-gamma2 ... -

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Basic information

Entry
Database: PDB / ID: 8g4n
TitleNative GABA-A receptor from the mouse brain, alpha1-beta2-gamma2 subtype, in complex with GABA, Zolpidem, and endogenous neurosteroids
Components
  • (Gamma-aminobutyric acid receptor subunit ...) x 3
  • Heavy Chain of 8E3 Fab
  • Light Chain of 8E3 Fab
KeywordsMEMBRANE PROTEIN / ligand-gated ion channel / cys-loop receptor / neurotransmitter receptor
Function / homology
Function and homology information


GABA receptor activation / diazepam binding / inhibitory synapse / GABA receptor complex / organic cyclic compound binding / inhibitory extracellular ligand-gated monoatomic ion channel activity / inner ear receptor cell development / GABA-gated chloride ion channel activity / cellular response to histamine / inhibitory synapse assembly ...GABA receptor activation / diazepam binding / inhibitory synapse / GABA receptor complex / organic cyclic compound binding / inhibitory extracellular ligand-gated monoatomic ion channel activity / inner ear receptor cell development / GABA-gated chloride ion channel activity / cellular response to histamine / inhibitory synapse assembly / GABA-A receptor activity / GABA-A receptor complex / innervation / neurotransmitter receptor activity / postsynaptic specialization membrane / gamma-aminobutyric acid signaling pathway / synaptic transmission, GABAergic / adult behavior / chloride channel activity / cochlea development / chloride channel complex / GABA-ergic synapse / neuron development / regulation of postsynaptic membrane potential / presynaptic active zone membrane / chloride transmembrane transport / dendrite membrane / ligand-gated monoatomic ion channel activity involved in regulation of presynaptic membrane potential / post-embryonic development / transmitter-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / cytoplasmic vesicle membrane / chemical synaptic transmission / cytoplasmic vesicle / postsynapse / neuron projection / axon / glutamatergic synapse / synapse / plasma membrane
Similarity search - Function
Gamma-aminobutyric-acid A receptor, gamma 2 subunit / Gamma-aminobutyric acid receptor subunit gamma-1/4 / : / Gamma-aminobutyric-acid A receptor, alpha 1 subunit / Gamma-aminobutyric-acid A receptor, beta subunit / : / Gamma-aminobutyric-acid A receptor, alpha subunit / Gamma-aminobutyric acid A receptor/Glycine receptor alpha / Neurotransmitter-gated ion-channel, conserved site / Neurotransmitter-gated ion-channels signature. ...Gamma-aminobutyric-acid A receptor, gamma 2 subunit / Gamma-aminobutyric acid receptor subunit gamma-1/4 / : / Gamma-aminobutyric-acid A receptor, alpha 1 subunit / Gamma-aminobutyric-acid A receptor, beta subunit / : / Gamma-aminobutyric-acid A receptor, alpha subunit / Gamma-aminobutyric acid A receptor/Glycine receptor alpha / Neurotransmitter-gated ion-channel, conserved site / Neurotransmitter-gated ion-channels signature. / Neurotransmitter-gated ion-channel transmembrane domain / Neurotransmitter-gated ion-channel transmembrane region / Neurotransmitter-gated ion-channel transmembrane domain superfamily / Neuronal acetylcholine receptor / Neurotransmitter-gated ion-channel / Neurotransmitter-gated ion-channel ligand-binding domain / Neurotransmitter-gated ion-channel ligand-binding domain superfamily / Neurotransmitter-gated ion-channel ligand binding domain
Similarity search - Domain/homology
GAMMA-AMINO-BUTANOIC ACID / DODECANE / N-OCTANE / Chem-PIO / Chem-POV / Zolpidem / allopregnanolone / Gamma-aminobutyric acid receptor subunit gamma-2 / Gamma-aminobutyric acid receptor subunit alpha-1 / Gamma-aminobutyric acid receptor subunit beta-2
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.67 Å
AuthorsSun, C. / Gouaux, E.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5R01GM100400-08 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Nature / Year: 2023
Title: Cryo-EM structures reveal native GABA receptor assemblies and pharmacology.
Authors: Chang Sun / Hongtao Zhu / Sarah Clark / Eric Gouaux /
Abstract: Type A γ-aminobutyric acid receptors (GABARs) are the principal inhibitory receptors in the brain and the target of a wide range of clinical agents, including anaesthetics, sedatives, hypnotics and ...Type A γ-aminobutyric acid receptors (GABARs) are the principal inhibitory receptors in the brain and the target of a wide range of clinical agents, including anaesthetics, sedatives, hypnotics and antidepressants. However, our understanding of GABAR pharmacology has been hindered by the vast number of pentameric assemblies that can be derived from 19 different subunits and the lack of structural knowledge of clinically relevant receptors. Here, we isolate native murine GABAR assemblies containing the widely expressed α1 subunit and elucidate their structures in complex with drugs used to treat insomnia (zolpidem (ZOL) and flurazepam) and postpartum depression (the neurosteroid allopregnanolone (APG)). Using cryo-electron microscopy (cryo-EM) analysis and single-molecule photobleaching experiments, we uncover three major structural populations in the brain: the canonical α1β2γ2 receptor containing two α1 subunits, and two assemblies containing one α1 and either an α2 or α3 subunit, in which the single α1-containing receptors feature a more compact arrangement between the transmembrane and extracellular domains. Interestingly, APG is bound at the transmembrane α/β subunit interface, even when not added to the sample, revealing an important role for endogenous neurosteroids in modulating native GABARs. Together with structurally engaged lipids, neurosteroids produce global conformational changes throughout the receptor that modify the ion channel pore and the binding sites for GABA and insomnia medications. Our data reveal the major α1-containing GABAR assemblies, bound with endogenous neurosteroid, thus defining a structural landscape from which subtype-specific drugs can be developed.
History
DepositionFeb 10, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 20, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 4, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Oct 18, 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 ..._atom_site.auth_atom_id / _atom_site.label_atom_id / _chem_comp_atom.atom_id / _chem_comp_bond.atom_id_1 / _chem_comp_bond.atom_id_2

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Gamma-aminobutyric acid receptor subunit alpha-1
B: Gamma-aminobutyric acid receptor subunit beta-2
C: Gamma-aminobutyric acid receptor subunit alpha-1
D: Gamma-aminobutyric acid receptor subunit gamma-2
E: Gamma-aminobutyric acid receptor subunit beta-2
H: Heavy Chain of 8E3 Fab
J: Heavy Chain of 8E3 Fab
K: Light Chain of 8E3 Fab
L: Light Chain of 8E3 Fab
hetero molecules


Theoretical massNumber of molelcules
Total (without water)390,77563
Polymers372,8969
Non-polymers17,87954
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Gamma-aminobutyric acid receptor subunit ... , 3 types, 5 molecules ACBED

#1: Protein Gamma-aminobutyric acid receptor subunit alpha-1 / GABA(A) receptor subunit alpha-1


Mass: 51818.516 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse) / Strain: C57BL/6 / References: UniProt: P62812
#2: Protein Gamma-aminobutyric acid receptor subunit beta-2 / GABA(A) receptor subunit beta-2


Mass: 59265.312 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse) / Strain: C57BL/6 / References: UniProt: P63137
#3: Protein Gamma-aminobutyric acid receptor subunit gamma-2 / GABA(A) receptor subunit gamma-2


Mass: 55160.281 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse) / Strain: C57BL/6 / References: UniProt: P22723

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Antibody , 2 types, 4 molecules HJKL

#4: Antibody Heavy Chain of 8E3 Fab


Mass: 24159.895 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Spodoptera frugiperda (fall armyworm)
#5: Antibody Light Chain of 8E3 Fab


Mass: 23624.080 Da / Num. of mol.: 2
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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Sugars , 4 types, 8 molecules

#6: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}}LINUCSPDB-CARE
#7: Polysaccharide
alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2- ...alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 910.823 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3[DManpa1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/3,5,4/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3/a4-b1_b4-c1_c3-d1_c6-e1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}[(6+1)][a-D-Manp]{}}}}}LINUCSPDB-CARE
#8: Polysaccharide alpha-D-mannopyranose-(1-2)-alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-3)-alpha-D- ...alpha-D-mannopyranose-(1-2)-alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-3)-alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 1235.105 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-2DManpa1-3[DManpa1-3DManpa1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/3,7,6/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3-3-3/a4-b1_b4-c1_c3-d1_c6-f1_d2-e1_f3-g1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{[(2+1)][a-D-Manp]{}}[(6+1)][a-D-Manp]{[(3+1)][a-D-Manp]{}}}}}}LINUCSPDB-CARE
#16: 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 / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
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 , 7 types, 46 molecules

#9: Chemical ChemComp-Y4B / allopregnanolone / 3beta-hydroxy-5beta,10alpha,14beta,17alpha-pregnan-20-one / Brexanolone


Mass: 318.493 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C21H34O2 / Feature type: SUBJECT OF INVESTIGATION
#10: Chemical ChemComp-PIO / [(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(oxidanyl)-4,5-diphosphonooxy-cyclohexyl]oxy-phosphoryl]oxy-propyl] octanoate / dioctanoyl l-alpha-phosphatidyl-d-myo-inositol 4,5-diphosphate


Mass: 746.566 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C25H49O19P3
#11: Chemical
ChemComp-D12 / DODECANE / Dodecane


Mass: 170.335 Da / Num. of mol.: 17 / Source method: obtained synthetically / Formula: C12H26
#12: Chemical
ChemComp-OCT / N-OCTANE / Octane


Mass: 114.229 Da / Num. of mol.: 17 / Source method: obtained synthetically / Formula: C8H18
#13: Chemical
ChemComp-POV / (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate / POPC / POPC


Mass: 760.076 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C42H82NO8P / Comment: phospholipid*YM
#14: Chemical ChemComp-ABU / GAMMA-AMINO-BUTANOIC ACID / GAMMA(AMINO)-BUTYRIC ACID / Γ-Aminobutyric acid


Mass: 103.120 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C4H9NO2 / Feature type: SUBJECT OF INVESTIGATION / Comment: neurotransmitter, inhibitor*YM
#15: Chemical ChemComp-R5R / Zolpidem / Zolpidem


Mass: 307.390 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C19H21N3O / Feature type: SUBJECT OF INVESTIGATION / Comment: medication*YM

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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

ComponentName: Native GABAA Receptor isolated from mouse brain containing two alpha1 subunits, in complex with 8E3-Fab, GABA, Zolpidem, and endogenous neurosteroid
Type: COMPLEX / Entity ID: #4-#5 / Source: NATURAL
Molecular weightValue: 0.35 MDa / Experimental value: NO
Source (natural)Organism: Mus musculus (house mouse) / Strain: C57BL/6 / Organ: Brain
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMSodium ChlorideNaClSodium chloride1
220 mMTrisTris-HClTris1
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse.
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2100 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM softwareName: cryoSPARC / Version: 3.3.2 / Category: 3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.67 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 124019 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00318723
ELECTRON MICROSCOPYf_angle_d0.54525281
ELECTRON MICROSCOPYf_dihedral_angle_d13.9617124
ELECTRON MICROSCOPYf_chiral_restr0.0432897
ELECTRON MICROSCOPYf_plane_restr0.0043001

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