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- PDB-7qna: Cryo-EM structure of human full-length alpha4beta3gamma2 GABA(A)R... -

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Basic information

Entry
Database: PDB / ID: 7qna
TitleCryo-EM structure of human full-length alpha4beta3gamma2 GABA(A)R in complex with GABA 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 / circadian sleep/wake cycle, REM sleep / reproductive behavior / hard palate development / cellular response to histamine / GABA receptor activation / inner ear receptor cell development / inhibitory synapse assembly / GABA-A receptor activity / GABA-gated chloride ion channel activity ...benzodiazepine receptor activity / circadian sleep/wake cycle, REM sleep / reproductive behavior / hard palate development / cellular response to histamine / GABA receptor activation / inner ear receptor cell development / inhibitory synapse assembly / GABA-A receptor activity / GABA-gated chloride ion channel activity / GABA-A receptor complex / innervation / response to anesthetic / postsynaptic specialization membrane / inhibitory postsynaptic potential / gamma-aminobutyric acid signaling pathway / synaptic transmission, GABAergic / cellular response to zinc ion / exploration behavior / chloride channel activity / motor behavior / roof of mouth development / Signaling by ERBB4 / cochlea development / social behavior / chloride channel complex / extracellular ligand-gated monoatomic ion channel activity / dendrite membrane / cytoplasmic vesicle membrane / chloride transmembrane transport / cerebellum development / central nervous system development / learning / transmitter-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / GABA-ergic synapse / memory / dendritic spine / postsynaptic membrane / postsynapse / response to xenobiotic stimulus / cell surface / signal transduction / identical protein binding / plasma membrane
Similarity search - Function
Gamma-aminobutyric-acid A receptor, alpha 4 subunit / Gamma-aminobutyric acid receptor subunit gamma-1/4 / Gamma-aminobutyric-acid A receptor, alpha subunit / : / Gamma-aminobutyric-acid A receptor, beta 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 receptor subunit gamma-1/4 / Gamma-aminobutyric-acid A receptor, alpha subunit / : / Gamma-aminobutyric-acid A receptor, beta 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 / Immunoglobulins / Immunoglobulin-like / Sandwich / Mainly Beta
Similarity search - Domain/homology
GAMMA-AMINO-BUTANOIC ACID / Gamma-aminobutyric acid receptor subunit gamma-3 / 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: 3 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
Revision 2.0Nov 15, 2023Group: Atomic model / Data collection / Category: atom_site / chem_comp_atom / chem_comp_bond / Item: _atom_site.auth_atom_id / _atom_site.label_atom_id
Revision 2.1Oct 9, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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 gamma-2
D: Gamma-aminobutyric acid receptor subunit beta-3
E: Gamma-aminobutyric acid receptor subunit beta-3
N: Nanobody Nb25
hetero molecules


Theoretical massNumber of molelcules
Total (without water)298,70816
Polymers293,2466
Non-polymers5,46210
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area37390 Å2
ΔGint-108 kcal/mol
Surface area68560 Å2

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Components

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

#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 gamma-2


Mass: 55666.809 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): HEK293S TetR / Production host: Homo sapiens (human) / References: UniProt: A0A1W2PQX1

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Antibody / Non-polymers , 2 types, 2 molecules N

#4: Antibody Nanobody Nb25


Mass: 13341.741 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)
#8: Chemical ChemComp-ABU / GAMMA-AMINO-BUTANOIC ACID / GAMMA(AMINO)-BUTYRIC ACID


Mass: 103.120 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H9NO2 / Feature type: SUBJECT OF INVESTIGATION

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Sugars , 3 types, 9 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: obtained synthetically
DescriptorTypeProgram
DManpa1-3[DManpa1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam 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
[][D-1-deoxy-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: obtained synthetically
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam 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
[][D-1-deoxy-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


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 2 / 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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Details

Has ligand of interestY
Has protein modificationY

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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 alpha4beta3gamma2 GABA(A)R in complex with GABA 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 chlorideNaCl1
250 mMHEPES1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 30 mA (EasyGlow) / Grid material: GOLD / Grid type: UltrAuFoil R1.2/1.3
VitrificationCryogen 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 FIELD / 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: 47.46 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
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
11RELION3.1classification
13REFMAC5model refinement
14PHENIX1.19.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 55567 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00215164
ELECTRON MICROSCOPYf_angle_d0.39520619
ELECTRON MICROSCOPYf_dihedral_angle_d8.9835543
ELECTRON MICROSCOPYf_chiral_restr0.0382398
ELECTRON MICROSCOPYf_plane_restr0.0032505

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