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- EMDB-4411: Cryo-EM structure of the human synaptic alpha1-beta3-gamma2 GABAA... -

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Basic information

Entry
Database: EMDB / ID: EMD-4411
TitleCryo-EM structure of the human synaptic alpha1-beta3-gamma2 GABAA receptor in complex with Megabody38 in a lipid nanodisc
Map data
SampleHuman alpha1-beta3-gamma2 GABA-A receptor in complex with Megabody38 and in lipid nanodiscs
  • Human alpha1-beta3-gamma2 GABA-A receptor
  • (Megabody38) x 2
  • (Gamma-aminobutyric acid receptor subunit ...) x 3
  • (ligand) x 5
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
Gamma-aminobutyric-acid A receptor, alpha subunit / Neurotransmitter-gated ion-channel ligand-binding domain superfamily / Neurotransmitter-gated ion-channel transmembrane domain superfamily / Neurotransmitter-gated ion-channel, conserved site / Neurotransmitter-gated ion-channel ligand-binding domain / Neurotransmitter-gated ion-channel / Neurotransmitter-gated ion-channel transmembrane domain / Gamma-aminobutyric acid A receptor/Glycine receptor alpha / Gamma-aminobutyric-acid A receptor, gamma 2 subunit / Gamma-aminobutyric-acid A receptor, gamma subunit ...Gamma-aminobutyric-acid A receptor, alpha subunit / Neurotransmitter-gated ion-channel ligand-binding domain superfamily / Neurotransmitter-gated ion-channel transmembrane domain superfamily / Neurotransmitter-gated ion-channel, conserved site / Neurotransmitter-gated ion-channel ligand-binding domain / Neurotransmitter-gated ion-channel / Neurotransmitter-gated ion-channel transmembrane domain / Gamma-aminobutyric acid A receptor/Glycine receptor alpha / Gamma-aminobutyric-acid A receptor, gamma 2 subunit / Gamma-aminobutyric-acid A receptor, gamma subunit / Gamma-aminobutyric-acid A receptor, alpha 1 subunit / Gamma-aminobutyric-acid A receptor, beta subunit
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsLaverty D / Desai R / Uchanski T / Masiulis S / Wojciech JS / Malinauskas T / Zivanov J / Pardon E / Steyaert J / Miller KW / Aricescu AR
Funding support United Kingdom, United States, Switzerland, 7 items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)MR/L009609/1 United Kingdom
Medical Research Council (United Kingdom)MC_UP_A025_1013 United Kingdom
Medical Research Council (United Kingdom)MC_UP_1201/15 United Kingdom
Cancer Research UKC20724/A14414 United Kingdom
Human Frontier Science ProgramRGP0065/2014 United Kingdom
National Institutes of Health/National Institute of General Medical SciencesGM 58448 United States
Swiss National Science Foundation168735 Switzerland
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.
History
DepositionNov 12, 2018-
Header (metadata) releaseDec 26, 2018-
Map releaseJan 2, 2019-
UpdateSep 11, 2019-
Current statusSep 11, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6i53
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_4411.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 256 pix.
= 273.92 Å
1.07 Å/pix.
x 256 pix.
= 273.92 Å
1.07 Å/pix.
x 256 pix.
= 273.92 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.04
Minimum - Maximum-0.14780992 - 0.22108346
Average (Standard dev.)0.00032399263 (±0.0058954824)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 273.92 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z273.920273.920273.920
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1480.2210.000

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Supplemental data

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Segmentation: #1

Fileemd_4411_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: #2

Fileemd_4411_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: #1

Fileemd_4411_half_map_2.map
Projections & Slices
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Sample components

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Entire Human alpha1-beta3-gamma2 GABA-A receptor in complex with Megabod...

EntireName: Human alpha1-beta3-gamma2 GABA-A receptor in complex with Megabody38 and in lipid nanodiscs
Number of components: 12

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Component #1: protein, Human alpha1-beta3-gamma2 GABA-A receptor in complex wit...

ProteinName: Human alpha1-beta3-gamma2 GABA-A receptor in complex with Megabody38 and in lipid nanodiscs
Recombinant expression: No
MassTheoretical: 400 kDa

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Component #2: protein, Human alpha1-beta3-gamma2 GABA-A receptor

ProteinName: Human alpha1-beta3-gamma2 GABA-A receptor / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human) / Strain: Embryonic kidney cells

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Component #3: protein, Megabody38

ProteinName: Megabody38 / Recombinant expression: No
SourceSpecies: Lama glama (llama)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #4: protein, Gamma-aminobutyric acid receptor subunit beta-3

ProteinName: Gamma-aminobutyric acid receptor subunit beta-3 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 54.444578 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human)

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Component #5: protein, Gamma-aminobutyric acid receptor subunit alpha-1

ProteinName: Gamma-aminobutyric acid receptor subunit alpha-1 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 52.916602 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human)

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Component #6: protein, Gamma-aminobutyric acid receptor subunit gamma-2

ProteinName: Gamma-aminobutyric acid receptor subunit gamma-2 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 56.922055 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human)

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Component #7: protein, Megabody38

ProteinName: Megabody38 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 13.462909 kDa
SourceSpecies: Lama glama (llama)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #8: ligand, N-ACETYL-D-GLUCOSAMINE

LigandName: N-ACETYL-D-GLUCOSAMINEN-Acetylglucosamine / Number of Copies: 14 / Recombinant expression: No
MassTheoretical: 0.221208 kDa

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Component #9: ligand, BETA-D-MANNOSE

LigandName: BETA-D-MANNOSE / Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 0.180156 kDa

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Component #10: ligand, ALPHA-D-MANNOSE

LigandName: ALPHA-D-MANNOSE / Number of Copies: 11 / Recombinant expression: No
MassTheoretical: 0.180156 kDa

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Component #11: ligand, (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propy...

LigandName: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate
Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 0.760076 kDa

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Component #12: ligand, [(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,...

LigandName: [(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
Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 0.746566 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.5 mg/mL / pH: 7.2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 287.15 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 30.84 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 75000.0 X (nominal), 75000.0 X (calibrated) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 500.0 - 700.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: FEI FALCON III (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 784

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 55449
3D reconstructionAlgorithm: FOURIER SPACE / Software: RELION
CTF correction: CTF parameters were estimated using GCTF and CTF correction performed in RELION (full phase and amplitude correction).
Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Modeling #1Refinement space: REAL

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