United Kingdom, Switzerland, United States, 7 items
Organization
Grant number
Country
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 Program
RGP0065/2014
United Kingdom
Cancer Research UK
C20724/A14414
United Kingdom
Swiss National Science Foundation
168735
Switzerland
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
GM 58448
United States
Citation
Journal: 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.
History
Deposition
Nov 12, 2018
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Header (metadata) release
Dec 26, 2018
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Map release
Jan 2, 2019
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Update
Mar 30, 2022
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Current status
Mar 30, 2022
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Supramolecule #1: Human alpha1-beta3-gamma2 GABA-A receptor in complex with Megabod...
Supramolecule
Name: Human alpha1-beta3-gamma2 GABA-A receptor in complex with Megabody38 and in lipid nanodiscs type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Molecular weight
Theoretical: 400 KDa
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Supramolecule #2: Human alpha1-beta3-gamma2 GABA-A receptor
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 287.15 K / Instrument: FEI VITROBOT MARK IV
Details
The purified receptor-nanodisc complex was mixed with Mb38 prior to grid preparation.
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Electron microscopy
Microscope
FEI TITAN KRIOS
Specialist optics
Phase plate: VOLTA PHASE PLATE
Image recording
Film or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 784 / Average exposure time: 60.0 sec. / Average electron dose: 30.84 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Software - Name: Gctf (ver. v1.18) Details: CTF parameters were estimated using GCTF and CTF correction performed in RELION (full phase and amplitude correction).
Final reconstruction
Number classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 55449
Initial angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Version: RELION
Final angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Version: RELION
Final 3D classification
Number classes: 6 / Software - Name: RELION (ver. v3.0)
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Atomic model buiding 1
Refinement
Space: REAL / Protocol: AB INITIO MODEL
Output model
PDB-6i53: Cryo-EM structure of the human synaptic alpha1-beta3-gamma2 GABAA receptor in complex with Megabody38 in a lipid nanodisc
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