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- PDB-6huk: CryoEM structure of human full-length alpha1beta3gamma2L GABA(A)R... -

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Basic information

Entry
Database: PDB / ID: 6huk
TitleCryoEM structure of human full-length alpha1beta3gamma2L GABA(A)R in complex with bicuculline and megabody Mb38.
Components
  • (Gamma-aminobutyric acid receptor subunit ...) x 3
  • Megabody Mb38
KeywordsMEMBRANE PROTEIN / GABAAR / Membrane / Channel / Nanobody / Megabody / Cys-loop / PLGIC / Inhibition / Signalling / CNS / Neurons / Chloride / Ion / GABA / BCC / Bicuculline / Antagonist
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 / inhibitory postsynaptic potential / chloride channel activity / 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
Neurotransmitter-gated ion-channel transmembrane region / Neurotransmitter-gated ion-channel transmembrane domain / Gamma-aminobutyric-acid A receptor, alpha subunit / Gamma-aminobutyric-acid A receptor, beta subunit / Gamma-aminobutyric-acid A receptor, alpha 1 subunit / Gamma-aminobutyric-acid A receptor, gamma subunit / Gamma-aminobutyric-acid A receptor, gamma 2 subunit / Gamma-aminobutyric acid A receptor/Glycine receptor alpha / Neurotransmitter-gated ion-channel / Neurotransmitter-gated ion-channel ligand-binding domain ...Neurotransmitter-gated ion-channel transmembrane region / Neurotransmitter-gated ion-channel transmembrane domain / Gamma-aminobutyric-acid A receptor, alpha subunit / Gamma-aminobutyric-acid A receptor, beta subunit / Gamma-aminobutyric-acid A receptor, alpha 1 subunit / Gamma-aminobutyric-acid A receptor, gamma subunit / Gamma-aminobutyric-acid A receptor, gamma 2 subunit / Gamma-aminobutyric acid A receptor/Glycine receptor alpha / Neurotransmitter-gated ion-channel / Neurotransmitter-gated ion-channel ligand-binding domain / Neurotransmitter-gated ion-channel, conserved site / Neurotransmitter-gated ion-channel transmembrane domain superfamily / Neurotransmitter-gated ion-channel ligand-binding domain superfamily / Neurotransmitter-gated ion-channel ligand binding domain
Gamma-aminobutyric acid receptor subunit alpha-1 / Gamma-aminobutyric acid receptor subunit alpha-1 / Gamma-aminobutyric acid receptor subunit gamma-2 / Gamma-aminobutyric acid receptor subunit beta-3
Biological speciesBos taurus (cattle)
Homo sapiens (human)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.69 Å
AuthorsMasiulis, S. / Desai, R. / Uchanski, T. / Serna Martin, I. / Laverty, D. / Karia, D. / Malinauskas, T. / Jasenko, Z. / Pardon, E. / Kotecha, A. / Steyaert, J. / Miller, K.W. / Aricescu, A.R.
Funding support United Kingdom, Switzerland, United States, 8items
OrganizationGrant numberCountry
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
Biotechnology and Biological Sciences Research CouncilBB/M024709/1 United Kingdom
Cancer Research UKC20724/A14414 United Kingdom
Human Frontier Science ProgramRGP0065/2014 United Kingdom
Swiss National Science Foundation168735 Switzerland
National Institutes of Health/National Institute of General Medical SciencesGM 58448 United States
CitationJournal: Nature / Year: 2019
Title: GABA receptor signalling mechanisms revealed by structural pharmacology.
Authors: Simonas Masiulis / Rooma Desai / Tomasz Uchański / Itziar Serna Martin / Duncan Laverty / Dimple Karia / Tomas Malinauskas / Jasenko Zivanov / Els Pardon / Abhay Kotecha / Jan Steyaert / ...Authors: Simonas Masiulis / Rooma Desai / Tomasz Uchański / Itziar Serna Martin / Duncan Laverty / Dimple Karia / Tomas Malinauskas / Jasenko Zivanov / Els Pardon / Abhay Kotecha / Jan Steyaert / Keith W Miller / A Radu Aricescu /
Abstract: Type-A γ-aminobutyric (GABA) receptors are ligand-gated chloride channels with a very rich pharmacology. Some of their modulators, including benzodiazepines and general anaesthetics, are among the ...Type-A γ-aminobutyric (GABA) receptors are ligand-gated chloride channels with a very rich pharmacology. Some of their modulators, including benzodiazepines and general anaesthetics, are among the most successful drugs in clinical use and are common substances of abuse. Without reliable structural data, the mechanistic basis for the pharmacological modulation of GABA receptors remains largely unknown. Here we report several high-resolution cryo-electron microscopy structures in which the full-length human α1β3γ2L GABA receptor in lipid nanodiscs is bound to the channel-blocker picrotoxin, the competitive antagonist bicuculline, the agonist GABA (γ-aminobutyric acid), and the classical benzodiazepines alprazolam and diazepam. We describe the binding modes and mechanistic effects of these ligands, the closed and desensitized states of the GABA receptor gating cycle, and the basis for allosteric coupling between the extracellular, agonist-binding region and the transmembrane, pore-forming region. This work provides a structural framework in which to integrate previous physiology and pharmacology research and a rational basis for the development of GABA receptor modulators.
Validation Report
SummaryFull reportAbout validation report
History
DepositionOct 8, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 2, 2019Provider: repository / Type: Initial release
Revision 1.1Jan 16, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jan 23, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_id_ISSN / _citation.journal_volume ..._citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Dec 11, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]

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Structure visualization

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Assembly

Deposited unit
A: Gamma-aminobutyric acid receptor subunit alpha-1,Gamma-aminobutyric acid receptor subunit alpha-1
B: Gamma-aminobutyric acid receptor subunit beta-3
C: Gamma-aminobutyric acid receptor subunit gamma-2
D: Gamma-aminobutyric acid receptor subunit alpha-1,Gamma-aminobutyric acid receptor subunit alpha-1
E: Gamma-aminobutyric acid receptor subunit beta-3
G: Megabody Mb38
hetero molecules


Theoretical massNumber of molelcules
Total (without water)337,63640
Polymers329,4296
Non-polymers8,20734
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area41150 Å2
ΔGint-96 kcal/mol
Surface area73010 Å2
MethodPISA

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Components

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

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


Mass: 52916.602 Da / Num. of mol.: 2
Details: Potential signal peptide: MKKSPGLSDY LWAWTLFLST LTGRSYG FLAG tag: DYKDDDDK,Potential signal peptide: MKKSPGLSDY LWAWTLFLST LTGRSYG FLAG tag: DYKDDDDK
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bos taurus (cattle), (gene. exp.) Homo sapiens (human)
Gene: GABRA1 / Cell line (production host): HEK293S / Production host: Homo sapiens (human) / References: UniProt: P08219, UniProt: P14867
#2: Protein Gamma-aminobutyric acid receptor subunit beta-3 / GABA(A) receptor subunit beta-3


Mass: 54444.578 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GABRB3 / Cell line (production host): HEK293S / Production host: Homo sapiens (human) / References: UniProt: P28472
#3: Protein Gamma-aminobutyric acid receptor subunit gamma-2 / GABA(A) receptor subunit gamma-2


Mass: 56922.055 Da / Num. of mol.: 1 / Details: Linker sequence: GGSGGSGGSGK 1D4 tag: TETSQVAPA
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GABRG2 / Cell line (production host): HEK293S / Production host: Homo sapiens (human) / References: UniProt: P18507

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Protein , 1 types, 1 molecules G

#4: Protein Megabody Mb38


Mass: 57784.301 Da / Num. of mol.: 1 / Details: 6His tag: HHHHHH EPEA tag: EPEA
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, 30 molecules

#7: Sugar
ChemComp-NAG / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine


Mass: 221.208 Da / Num. of mol.: 14
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
#8: Sugar
ChemComp-BMA / BETA-D-MANNOSE / Mannose


Mass: 180.156 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Formula: C6H12O6
#9: Sugar
ChemComp-MAN / ALPHA-D-MANNOSE / Mannose


Mass: 180.156 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Formula: C6H12O6

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Non-polymers , 2 types, 4 molecules

#5: Chemical ChemComp-H0Z / bicuculline methochloride / Bicuculline


Mass: 367.352 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C20H17NO6 / Feature type: SUBJECT OF INVESTIGATION / Comment: antagonist, alkaloid*YM
#6: Chemical ChemComp-PIO / [(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 / dioctanoyl l-alpha-phosphatidyl-d-myo-inositol 4,5-diphosphate


Mass: 746.566 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C25H49O19P3

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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

Component
IDNameTypeEntity IDParent-IDSource
1Human full-length heteromeric alpha1beta3gamma2L GABA(A)R in complex with bicuculline and megabody Mb38.COMPLEX1, 2, 3, 40MULTIPLE SOURCES
2Human full-length heteromeric alpha1beta3gamma2L GABA(A)RCOMPLEX1,2,31RECOMBINANT
3megabody Mb38COMPLEX41RECOMBINANT
Molecular weightValue: 0.33 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDCellular locationOrgan
22Homo sapiens (human)9606Plasma membraneBrain
33Lama glama (llama)9844
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCell
22Homo sapiens (human)9606HEK293S
33Esherichia coli562
Buffer solutionpH: 7.6
SpecimenConc.: 0.1 mg/ml / Details: Monodisperse sample / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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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: 75000 X / Nominal defocus max: 700 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 30 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)
EM imaging opticsPhase plate: Volta phase plate, FEI company.

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Processing

EM software
IDNameVersionCategoryDetails
2EPUimage acquisition
4Gctf1.08CTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10RELION3initial Euler assignmentRefine3D
11RELION3final Euler assignmentRefine3D
12RELION3classificationClass3D
13RELION33D reconstructionRefine3D
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 489434
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.69 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 30536 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT

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