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- PDB-6w2y: CryoEM Structure of GABAB1b Homodimer -

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Basic information

Entry
Database: PDB / ID: 6w2y
TitleCryoEM Structure of GABAB1b Homodimer
ComponentsGamma-aminobutyric acid type B receptor subunit 1
KeywordsSIGNALING PROTEIN / Inhibitor / Homodimer / GPCR
Function / homology
Function and homology information


G protein-coupled neurotransmitter receptor activity involved in regulation of postsynaptic membrane potential / neuron-glial cell signaling / G protein-coupled receptor heterodimeric complex / G protein-coupled GABA receptor activity / gamma-aminobutyric acid signaling pathway / GABA-ergic synapse / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / Schaffer collateral - CA1 synapse / presynaptic membrane / transmembrane signaling receptor activity ...G protein-coupled neurotransmitter receptor activity involved in regulation of postsynaptic membrane potential / neuron-glial cell signaling / G protein-coupled receptor heterodimeric complex / G protein-coupled GABA receptor activity / gamma-aminobutyric acid signaling pathway / GABA-ergic synapse / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / Schaffer collateral - CA1 synapse / presynaptic membrane / transmembrane signaling receptor activity / postsynaptic membrane / G protein-coupled receptor signaling pathway / dendrite / integral component of plasma membrane / extracellular region / plasma membrane / cytoplasm
Sushi/SCR/CCP superfamily / Periplasmic binding protein-like I / GPCR family 3, C-terminal / GPCR family 3, GABA-B receptor / Receptor, ligand binding region / Sushi/SCR/CCP domain
Gamma-aminobutyric acid type B receptor subunit 1
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsPapasergi-Scott, M.M. / Robertson, M.J. / Skiniotis, G.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)1R01NS092695-01 United States
CitationJournal: Nature / Year: 2020
Title: Structures of metabotropic GABA receptor.
Authors: Makaía M Papasergi-Scott / Michael J Robertson / Alpay B Seven / Ouliana Panova / Jesper M Mathiesen / Georgios Skiniotis /
Abstract: GABA (γ-aminobutyric acid) stimulation of the metabotropic GABA receptor results in prolonged inhibition of neurotransmission that is central to brain physiology. GABA belongs to the Family C of G ...GABA (γ-aminobutyric acid) stimulation of the metabotropic GABA receptor results in prolonged inhibition of neurotransmission that is central to brain physiology. GABA belongs to the Family C of G protein-coupled receptors (GPCRs), which operate as dimers to relay synaptic neurotransmitter signals into a cellular response through the binding and activation of heterotrimeric G proteins. GABA, however, is unique in its function as an obligate heterodimer in which agonist binding and G protein activation take place on distinct subunits. Here we show structures of heterodimeric and homodimeric full-length GABA receptors. Complemented by cellular signaling assays and atomistic simulations, the structures reveal an essential role for the GABA extracellular loop 2 (ECL2) in relaying structural transitions by ordering the linker connecting the extracellular ligand-binding domain to the transmembrane region. Furthermore, the ECL2 of both GABA subunits caps and interacts with the hydrophilic head of a phospholipid occupying the extracellular half of the transmembrane domain, thereby providing a potentially crucial link between ligand binding and the receptor core that engages G protein. These results provide a starting framework to decipher mechanistic modes of signal transduction mediated by GABA dimers and have important implications for rational drug design targeting these receptors.
Validation Report
SummaryFull reportAbout validation report
History
DepositionMar 8, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 1, 2020Provider: repository / Type: Initial release

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Assembly

Deposited unit
A: Gamma-aminobutyric acid type B receptor subunit 1
B: Gamma-aminobutyric acid type B receptor subunit 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)192,46216
Polymers188,3472
Non-polymers4,11514
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area5150 Å2
ΔGint-20 kcal/mol
Surface area53910 Å2

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Components

#1: Protein Gamma-aminobutyric acid type B receptor subunit 1 / Gb1


Mass: 94173.570 Da / Num. of mol.: 2 / Fragment: UNP residues 30-844
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GABBR1, GPRC3A / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9UBS5
#2: Sugar
ChemComp-NAG / N-ACETYL-D-GLUCOSAMINE


Mass: 221.208 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C8H15NO6
#3: Chemical ChemComp-MG / MAGNESIUM ION / Magnesium


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#4: Chemical ChemComp-L9Q / (1S)-2-{[(S)-(2-aminoethoxy)(hydroxy)phosphoryl]oxy}-1-[(octadecanoyloxy)methyl]ethyl (9Z)-octadec-9-enoate / 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine


Mass: 746.050 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C41H80NO8P
#5: Chemical ChemComp-SGG / [(2~{S})-3-[[(1~{S})-1-(3,4-dichlorophenyl)ethyl]amino]-2-oxidanyl-propyl]-(phenylmethyl)phosphinic acid


Mass: 402.252 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C18H22Cl2NO3P
Has ligand of interestN

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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: GABAB1b Homodimer / Type: COMPLEX / Entity ID: 1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2150 mMsodium chlorideNaClSodium chloride1
30.004 %Glyco-diosgenin (GDN)C56H92O251
40.0004 %Cholesterol hemisuccinateC31H50O41
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 291 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / C2 aperture diameter: 50 µm
Image recordingAverage exposure time: 8 sec. / Electron dose: 49.7 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansMovie frames/image: 40

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
4RELION3CTF correction
10RELION3initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 282811 / Symmetry type: POINT

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