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- PDB-7qnc: Cryo-EM structure of human full-length extrasynaptic alpha4beta3d... -

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Basic information

Entry
Database: PDB / ID: 7qnc
TitleCryo-EM structure of human full-length extrasynaptic alpha4beta3delta GABA(A)R in complex with THIP (gaboxadol), histamine and nanobody Nb25
Components
  • (Gamma-aminobutyric acid receptor subunit ...) x 3
  • Nanobody Nb25
KeywordsMEMBRANE PROTEIN / pentameric ligand-gated ion channel / neurotransmitter receptor / GABA receptor
Function / homology
Function and homology information


benzodiazepine receptor activity / inhibitory extracellular ligand-gated monoatomic ion channel activity / GABA receptor activation / GABA-gated chloride ion channel activity / cellular response to histamine / inhibitory synapse assembly / GABA-A receptor activity / GABA-A receptor complex / postsynaptic specialization membrane / neurotransmitter receptor activity ...benzodiazepine receptor activity / inhibitory extracellular ligand-gated monoatomic ion channel activity / GABA receptor activation / GABA-gated chloride ion channel activity / cellular response to histamine / inhibitory synapse assembly / GABA-A receptor activity / GABA-A receptor complex / postsynaptic specialization membrane / neurotransmitter receptor activity / synaptic transmission, GABAergic / gamma-aminobutyric acid signaling pathway / chloride channel activity / roof of mouth development / Signaling by ERBB4 / chloride channel complex / GABA-ergic synapse / regulation of postsynaptic membrane potential / chloride transmembrane transport / dendrite membrane / transmitter-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / cytoplasmic vesicle membrane / postsynapse / postsynaptic membrane / neuron projection / axon / neuronal cell body / dendrite / synapse / signal transduction / identical protein binding / plasma membrane
Similarity search - Function
Gamma-aminobutyric-acid A receptor, alpha 4 subunit / Gamma-aminobutyric-acid A receptor delta 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 ...Gamma-aminobutyric-acid A receptor, alpha 4 subunit / Gamma-aminobutyric-acid A receptor delta 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
DECANE / Chem-EI7 / HISTAMINE / Gamma-aminobutyric acid receptor subunit delta / Gamma-aminobutyric acid receptor subunit beta-3 / Gamma-aminobutyric acid receptor subunit alpha-4
Similarity search - Component
Biological speciesHomo sapiens (human)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsSente, A. / Desai, R. / Naydenova, K. / Malinauskas, T. / Jounaidi, Y. / Miehling, J. / Zhou, X. / Masiulis, S. / Hardwick, S.W. / Chirgadze, D.Y. ...Sente, A. / Desai, R. / Naydenova, K. / Malinauskas, T. / Jounaidi, Y. / Miehling, J. / Zhou, X. / Masiulis, S. / Hardwick, S.W. / Chirgadze, D.Y. / Miller, K.W. / Aricescu, A.R.
Funding support United Kingdom, 5items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MR/L009609/1 United Kingdom
Medical Research Council (MRC, United Kingdom)MC_UP_1201/15 United Kingdom
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1R01-GM135550 United Kingdom
Wellcome Trust206171/Z/17/Z United Kingdom
Wellcome Trust202905/Z/16/Z United Kingdom
CitationJournal: Nature / Year: 2022
Title: Differential assembly diversifies GABA receptor structures and signalling.
Authors: Andrija Sente / Rooma Desai / Katerina Naydenova / Tomas Malinauskas / Youssef Jounaidi / Jonas Miehling / Xiaojuan Zhou / Simonas Masiulis / Steven W Hardwick / Dimitri Y Chirgadze / Keith ...Authors: Andrija Sente / Rooma Desai / Katerina Naydenova / Tomas Malinauskas / Youssef Jounaidi / Jonas Miehling / Xiaojuan Zhou / Simonas Masiulis / Steven W Hardwick / Dimitri Y Chirgadze / Keith W Miller / A Radu Aricescu /
Abstract: Type A γ-aminobutyric acid receptors (GABARs) are pentameric ligand-gated chloride channels that mediate fast inhibitory signalling in neural circuits and can be modulated by essential medicines ...Type A γ-aminobutyric acid receptors (GABARs) are pentameric ligand-gated chloride channels that mediate fast inhibitory signalling in neural circuits and can be modulated by essential medicines including general anaesthetics and benzodiazepines. Human GABAR subunits are encoded by 19 paralogous genes that can, in theory, give rise to 495,235 receptor types. However, the principles that govern the formation of pentamers, the permutational landscape of receptors that may emerge from a subunit set and the effect that this has on GABAergic signalling remain largely unknown. Here we use cryogenic electron microscopy to determine the structures of extrasynaptic GABARs assembled from α4, β3 and δ subunits, and their counterparts incorporating γ2 instead of δ subunits. In each case, we identified two receptor subtypes with distinct stoichiometries and arrangements, all four differing from those previously observed for synaptic, α1-containing receptors. This, in turn, affects receptor responses to physiological and synthetic modulators by creating or eliminating ligand-binding sites at subunit interfaces. We provide structural and functional evidence that selected GABAR arrangements can act as coincidence detectors, simultaneously responding to two neurotransmitters: GABA and histamine. Using assembly simulations and single-cell RNA sequencing data, we calculated the upper bounds for receptor diversity in recombinant systems and in vivo. We propose that differential assembly is a pervasive mechanism for regulating the physiology and pharmacology of GABARs.
History
DepositionDec 20, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 13, 2022Provider: repository / Type: Initial release
Revision 1.1Apr 20, 2022Group: 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

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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-4
B: Gamma-aminobutyric acid receptor subunit beta-3
C: Gamma-aminobutyric acid receptor subunit beta-3
D: Gamma-aminobutyric acid receptor subunit beta-3
E: Gamma-aminobutyric acid receptor subunit delta
F: Nanobody Nb25
G: Nanobody Nb25
hetero molecules


Theoretical massNumber of molelcules
Total (without water)310,53227
Polymers303,3587
Non-polymers7,17320
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area40150 Å2
ΔGint-51 kcal/mol
Surface area74520 Å2

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Components

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

#1: Protein Gamma-aminobutyric acid receptor subunit alpha-4 / GABA(A) receptor subunit alpha-4


Mass: 61696.047 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GABRA4 / Cell line (production host): HEK293S TetR / Production host: Homo sapiens (human) / References: UniProt: P48169
#2: Protein Gamma-aminobutyric acid receptor subunit beta-3 / GABA(A) receptor subunit beta-3


Mass: 54180.348 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GABRB3 / Cell line (production host): HEK293S TetR / Production host: Homo sapiens (human) / References: UniProt: P28472
#3: Protein Gamma-aminobutyric acid receptor subunit delta / GABA(A) receptor subunit delta


Mass: 52437.926 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GABRD / Cell line (production host): HEK293S TetR / Production host: Homo sapiens (human) / References: UniProt: O14764

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Antibody , 1 types, 2 molecules FG

#4: Antibody Nanobody Nb25


Mass: 13341.741 Da / Num. of mol.: 2
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, 13 molecules

#5: 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.: 4
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
#6: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#7: 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.: 6 / Source method: obtained synthetically / 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 , 3 types, 7 molecules

#8: Chemical ChemComp-D10 / DECANE / Decane


Mass: 142.282 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H22
#9: Chemical ChemComp-EI7 / 4,5,6,7-tetrahydro-[1,2]oxazolo[5,4-c]pyridin-3-one / gaboxadol / Gaboxadol


Mass: 140.140 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C6H8N2O2 / Feature type: SUBJECT OF INVESTIGATION / Comment: alkaloid*YM
#10: Chemical ChemComp-HSM / HISTAMINE / Histamine


Mass: 111.145 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C5H9N3 / Feature type: SUBJECT OF INVESTIGATION / Comment: neurotransmitter, hormone*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: Human full-length extrasynaptic alpha4beta3delta GABA(A)R in complex with THIP (gaboxadol), histamine and nanobody Nb25
Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293S TetR
Buffer solutionpH: 7.6
Buffer component
IDConc.NameFormulaBuffer-ID
1300 mMSodium chlorideNaClSodium chloride1
250 mMHEPES1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: LEICA PLUNGER / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 287 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: 130000 X / Nominal defocus max: 2100 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4CTFFIND4.1.13CTF correction
7Cootmodel fitting
9REFMAC5model refinement
10PHENIX1.19.2model refinement
11RELION3.1initial Euler assignment
12RELION3.1final Euler assignment
13RELION3.1classification
14RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 61854 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00216291
ELECTRON MICROSCOPYf_angle_d0.48622160
ELECTRON MICROSCOPYf_dihedral_angle_d10.9225981
ELECTRON MICROSCOPYf_chiral_restr0.0412548
ELECTRON MICROSCOPYf_plane_restr0.0052731

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