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- PDB-6i53: Cryo-EM structure of the human synaptic alpha1-beta3-gamma2 GABAA... -

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Entry
Database: PDB / ID: 6i53
TitleCryo-EM structure of the human synaptic alpha1-beta3-gamma2 GABAA receptor in complex with Megabody38 in a lipid nanodisc
Components
  • (Gamma-aminobutyric acid receptor subunit ...) x 3
  • Megabody38
KeywordsTRANSPORT PROTEIN / Ligand gated ion channel Cys-Loop receptor GABA-A receptor
Function / homology
Function and homology information


benzodiazepine receptor activity / inner ear receptor cell development / inhibitory extracellular ligand-gated ion channel activity / GABA-A receptor complex / GABA-A receptor activity / GABA-gated chloride ion channel activity / GABA receptor complex / inhibitory synapse assembly / cellular response to histamine / synaptic transmission, GABAergic ...benzodiazepine receptor activity / inner ear receptor cell development / inhibitory extracellular ligand-gated ion channel activity / GABA-A receptor complex / GABA-A receptor activity / GABA-gated chloride ion channel activity / GABA receptor complex / inhibitory synapse assembly / cellular response to histamine / synaptic transmission, GABAergic / gamma-aminobutyric acid signaling pathway / integral component of postsynaptic specialization membrane / innervation / regulation of postsynaptic membrane potential / chloride channel activity / inhibitory postsynaptic potential / chloride channel complex / chloride transmembrane transport / adult behavior / dendrite membrane / cochlea development / GABA-ergic synapse / nervous system process / roof of mouth development / transmitter-gated ion channel activity involved in regulation of postsynaptic membrane potential / ion transmembrane transport / post-embryonic development / regulation of membrane potential / sensory perception of sound / cytoplasmic vesicle membrane / postsynapse / postsynaptic membrane / chemical synaptic transmission / drug binding / negative regulation of neuron apoptotic process / neuron projection / synapse / cell junction / axon / signal transduction / integral component of plasma membrane / identical protein binding / plasma membrane
Neurotransmitter-gated ion-channel transmembrane region / Neurotransmitter-gated ion-channel transmembrane domain / Gamma-aminobutyric-acid A receptor, alpha subunit / Gamma-aminobutyric-acid A receptor, beta subunit / Gamma-aminobutyric-acid A receptor, alpha 1 subunit / Gamma-aminobutyric-acid A receptor, gamma subunit / Gamma-aminobutyric-acid A receptor, gamma 2 subunit / Gamma-aminobutyric acid A receptor/Glycine receptor alpha / Neurotransmitter-gated ion-channel / Neurotransmitter-gated ion-channel ligand-binding domain ...Neurotransmitter-gated ion-channel transmembrane region / Neurotransmitter-gated ion-channel transmembrane domain / Gamma-aminobutyric-acid A receptor, alpha subunit / Gamma-aminobutyric-acid A receptor, beta subunit / Gamma-aminobutyric-acid A receptor, alpha 1 subunit / Gamma-aminobutyric-acid A receptor, gamma subunit / Gamma-aminobutyric-acid A receptor, gamma 2 subunit / Gamma-aminobutyric acid A receptor/Glycine receptor alpha / Neurotransmitter-gated ion-channel / Neurotransmitter-gated ion-channel ligand-binding domain / Neurotransmitter-gated ion-channel, conserved site / Neurotransmitter-gated ion-channel transmembrane domain superfamily / Neurotransmitter-gated ion-channel ligand-binding domain superfamily / Neurotransmitter-gated ion-channel ligand binding domain
Gamma-aminobutyric acid receptor subunit alpha-1 / Gamma-aminobutyric acid receptor subunit gamma-2 / Gamma-aminobutyric acid receptor subunit beta-3
Biological speciesHomo sapiens (human)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsLaverty, D. / Desai, R. / Uchanski, T. / Masiulis, S. / Wojciech, J.S. / Malinauskas, T. / Zivanov, J. / Pardon, E. / Steyaert, J. / Miller, K.W. / Aricescu, A.R.
Funding support United Kingdom, Switzerland, United States, 7items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)MR/L009609/1 United Kingdom
Medical Research Council (United Kingdom)MC_UP_1201/15 United Kingdom
Medical Research Council (United Kingdom)MC_UP_A025_1013 United Kingdom
Human Frontier Science ProgramRGP0065/2014 United Kingdom
Cancer Research UKC20724/A14414 United Kingdom
Swiss National Science Foundation168735 Switzerland
National Institutes of Health/National Institute of General Medical SciencesGM 58448 United States
CitationJournal: Nature / Year: 2019
Title: Cryo-EM structure of the human α1β3γ2 GABA receptor in a lipid bilayer.
Authors: Duncan Laverty / Rooma Desai / Tomasz Uchański / Simonas Masiulis / Wojciech J Stec / Tomas Malinauskas / Jasenko Zivanov / Els Pardon / Jan Steyaert / Keith W Miller / A Radu Aricescu /
Abstract: Type A γ-aminobutyric acid (GABA) receptors are pentameric ligand-gated ion channels and the main drivers of fast inhibitory neurotransmission in the vertebrate nervous system. Their dysfunction is ...Type A γ-aminobutyric acid (GABA) receptors are pentameric ligand-gated ion channels and the main drivers of fast inhibitory neurotransmission in the vertebrate nervous system. Their dysfunction is implicated in a range of neurological disorders, including depression, epilepsy and schizophrenia. Among the numerous assemblies that are theoretically possible, the most prevalent in the brain are the α1β2/3γ2 GABA receptors. The β3 subunit has an important role in maintaining inhibitory tone, and the expression of this subunit alone is sufficient to rescue inhibitory synaptic transmission in β1-β3 triple knockout neurons. So far, efforts to generate accurate structural models for heteromeric GABA receptors have been hampered by the use of engineered receptors and the presence of detergents. Notably, some recent cryo-electron microscopy reconstructions have reported 'collapsed' conformations; however, these disagree with the structure of the prototypical pentameric ligand-gated ion channel the Torpedo nicotinic acetylcholine receptor, the large body of structural work on homologous homopentameric receptor variants and the logic of an ion-channel architecture. Here we present a high-resolution cryo-electron microscopy structure of the full-length human α1β3γ2L-a major synaptic GABA receptor isoform-that is functionally reconstituted in lipid nanodiscs. The receptor is bound to a positive allosteric modulator 'megabody' and is in a desensitized conformation. Each GABA receptor pentamer contains two phosphatidylinositol-4,5-bisphosphate molecules, the head groups of which occupy positively charged pockets in the intracellular juxtamembrane regions of α1 subunits. Beyond this level, the intracellular M3-M4 loops are largely disordered, possibly because interacting post-synaptic proteins are not present. This structure illustrates the molecular principles of heteromeric GABA receptor organization and provides a reference framework for future mechanistic investigations of GABAergic signalling and pharmacology.
Validation Report
SummaryFull reportAbout validation report
History
DepositionNov 12, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 2, 2019Provider: repository / Type: Initial release
Revision 1.1Jan 16, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Jan 23, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_id_ISSN / _citation.journal_volume ..._citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Aug 21, 2019Group: Data collection / Experimental preparation / Category: em_sample_support / Item: _em_sample_support.grid_type
Revision 1.4Sep 11, 2019Group: Data collection / Source and taxonomy / Category: entity_src_gen / Item: _entity_src_gen.pdbx_end_seq_num

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Structure visualization

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Assembly

Deposited unit
E: Gamma-aminobutyric acid receptor subunit beta-3
A: Gamma-aminobutyric acid receptor subunit alpha-1
B: Gamma-aminobutyric acid receptor subunit beta-3
C: Gamma-aminobutyric acid receptor subunit gamma-2
D: Gamma-aminobutyric acid receptor subunit alpha-1
G: Megabody38
hetero molecules


Theoretical massNumber of molelcules
Total (without water)294,68040
Polymers285,1076
Non-polymers9,57334
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area40550 Å2
ΔGint-84 kcal/mol
Surface area72780 Å2
MethodPISA

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Components

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

#1: Protein Gamma-aminobutyric acid receptor subunit beta-3 / GABA(A) receptor subunit beta-3


Mass: 54444.578 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GABRB3 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: P28472
#2: Protein Gamma-aminobutyric acid receptor subunit alpha-1 / GABA(A) receptor subunit alpha-1


Mass: 52916.602 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GABRA1 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: P14867
#3: Protein Gamma-aminobutyric acid receptor subunit gamma-2 / GABA(A) receptor subunit gamma-2


Mass: 56922.055 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GABRG2 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: P18507

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Protein , 1 types, 1 molecules G

#4: Protein Megabody38


Mass: 13462.909 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)

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Sugars , 3 types, 29 molecules

#5: Sugar
ChemComp-NAG / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine


Mass: 221.208 Da / Num. of mol.: 14
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
#6: Sugar
ChemComp-BMA / BETA-D-MANNOSE / Mannose


Mass: 180.156 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Formula: C6H12O6
#7: Sugar
ChemComp-MAN / ALPHA-D-MANNOSE / Mannose


Mass: 180.156 Da / Num. of mol.: 11
Source method: isolated from a genetically manipulated source
Formula: C6H12O6

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Non-polymers , 2 types, 5 molecules

#8: 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.: 3 / Source method: obtained synthetically / Formula: C42H82NO8P / Comment: phospholipid*YM
#9: 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: isolated from a genetically manipulated source
Formula: C25H49O19P3

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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
IDNameTypeEntity IDParent-IDSource
1Human alpha1-beta3-gamma2 GABA-A receptor in complex with Megabody38 and in lipid nanodiscsCOMPLEX1,2,3,40MULTIPLE SOURCES
2Human alpha1-beta3-gamma2 GABA-A receptorCOMPLEX1,2,31RECOMBINANT
3Megabody38COMPLEX41RECOMBINANT
Molecular weightValue: 0.4 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Lama glama (llama)9844
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
22Homo sapiens (human)9606Embryonic kidney cells
33Escherichia coli (E. coli)562
Buffer solutionpH: 7.2
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMSodium chlorideNaClSodium chloride1
210 mMHEPESC8H18N2O4S1
SpecimenConc.: 0.5 mg/ml
Details: The purified receptor-nanodisc complex was mixed with Mb38 prior to grid preparation.
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: Quantifoil, UltrAuFoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 287.15 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 magnification: 75000 X / Calibrated magnification: 75000 X / Nominal defocus max: 700 nm / Nominal defocus min: 500 nm / Calibrated defocus min: 500 nm / Calibrated defocus max: 700 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderModel: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 60 sec. / Electron dose: 30.84 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 784
EM imaging opticsPhase plate: Volta phase plate

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Processing

SoftwareName: PHENIX / Version: 1.14rc2_3191: / Classification: refinement
EM software
IDNameVersionCategory
5Gctfv1.18CTF correction
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
12RELIONv3.0classification
13RELION33D reconstruction
14PHENIXmodel refinement
CTF correctionDetails: CTF parameters were estimated using GCTF and CTF correction performed in RELION (full phase and amplitude correction).
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 55449 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
RefinementStereochemistry target values: CDL v1.2
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004915782
ELECTRON MICROSCOPYf_angle_d0.754321490
ELECTRON MICROSCOPYf_chiral_restr0.04952487
ELECTRON MICROSCOPYf_plane_restr0.00652605
ELECTRON MICROSCOPYf_dihedral_angle_d7.53989236

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