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- PDB-6x3w: Human GABAA receptor alpha1-beta2-gamma2 subtype in complex with ... -

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Basic information

Entry
Database: PDB / ID: 6x3w
TitleHuman GABAA receptor alpha1-beta2-gamma2 subtype in complex with GABA plus phenobarbital
Components
  • (Gamma-aminobutyric acid receptor subunit ...) x 3
  • IgG2b Fab Heavy Chain
  • Kappa Fab Light Chain
KeywordsMEMBRANE PROTEIN / Ion channel / Cys-loop receptor / pentametic ligand gated channel / GABAA receptor
Function / homology
Function and homology information


benzodiazepine receptor activity / GABA receptor complex / inner ear receptor cell development / cellular response to histamine / GABA receptor activation / GABA-A receptor activity / GABA-gated chloride ion channel activity / GABA-A receptor complex / inhibitory synapse assembly / innervation ...benzodiazepine receptor activity / GABA receptor complex / inner ear receptor cell development / cellular response to histamine / GABA receptor activation / GABA-A receptor activity / GABA-gated chloride ion channel activity / GABA-A receptor complex / inhibitory synapse assembly / innervation / synaptic transmission, GABAergic / gamma-aminobutyric acid signaling pathway / postsynaptic specialization membrane / neurotransmitter receptor activity / chloride channel activity / cochlea development / adult behavior / Signaling by ERBB4 / chloride channel complex / regulation of postsynaptic membrane potential / transmembrane transporter complex / GABA-ergic synapse / chloride transmembrane transport / dendrite membrane / post-embryonic development / transmitter-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / cytoplasmic vesicle membrane / chemical synaptic transmission / postsynaptic membrane / postsynapse / dendritic spine / neuron projection / axon / synapse / extracellular exosome / plasma membrane
Similarity search - Function
Gamma-aminobutyric-acid A receptor, gamma 2 subunit / Gamma-aminobutyric acid receptor subunit gamma-1/4 / Gamma-aminobutyric-acid A receptor, alpha 1 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. ...Gamma-aminobutyric-acid A receptor, gamma 2 subunit / Gamma-aminobutyric acid receptor subunit gamma-1/4 / Gamma-aminobutyric-acid A receptor, alpha 1 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
GAMMA-AMINO-BUTANOIC ACID / 5-ethyl-5-phenylpyrimidine-2,4,6(1H,3H,5H)-trione / Gamma-aminobutyric acid receptor subunit alpha-1 / Gamma-aminobutyric acid receptor subunit gamma-2 / Gamma-aminobutyric acid receptor subunit beta-2
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsKim, J.J. / Gharpure, A. / Teng, J. / Zhuang, Y. / Howard, R.J. / Zhu, S. / Noviello, C.M. / Walsh, R.M. / Lindahl, E. / Hibbs, R.E.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Institute on Drug Abuse (NIH/NIDA)DA037492 United States
National Institutes of Health/National Institute on Drug Abuse (NIH/NIDA)DA042072 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS095899 United States
Welch FoundationI-1812 United States
American Heart Association20POST35200127 United States
CitationJournal: Nature / Year: 2020
Title: Shared structural mechanisms of general anaesthetics and benzodiazepines.
Authors: Jeong Joo Kim / Anant Gharpure / Jinfeng Teng / Yuxuan Zhuang / Rebecca J Howard / Shaotong Zhu / Colleen M Noviello / Richard M Walsh / Erik Lindahl / Ryan E Hibbs /
Abstract: Most general anaesthetics and classical benzodiazepine drugs act through positive modulation of γ-aminobutyric acid type A (GABA) receptors to dampen neuronal activity in the brain. However, direct ...Most general anaesthetics and classical benzodiazepine drugs act through positive modulation of γ-aminobutyric acid type A (GABA) receptors to dampen neuronal activity in the brain. However, direct structural information on the mechanisms of general anaesthetics at their physiological receptor sites is lacking. Here we present cryo-electron microscopy structures of GABA receptors bound to intravenous anaesthetics, benzodiazepines and inhibitory modulators. These structures were solved in a lipidic environment and are complemented by electrophysiology and molecular dynamics simulations. Structures of GABA receptors in complex with the anaesthetics phenobarbital, etomidate and propofol reveal both distinct and common transmembrane binding sites, which are shared in part by the benzodiazepine drug diazepam. Structures in which GABA receptors are bound by benzodiazepine-site ligands identify an additional membrane binding site for diazepam and suggest an allosteric mechanism for anaesthetic reversal by flumazenil. This study provides a foundation for understanding how pharmacologically diverse and clinically essential drugs act through overlapping and distinct mechanisms to potentiate inhibitory signalling in the brain.
History
DepositionMay 21, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 9, 2020Provider: repository / Type: Initial release
Revision 1.1Sep 16, 2020Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Sep 23, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 2.0Nov 15, 2023Group: Atomic model / Data collection / Database references
Category: atom_site / chem_comp_atom ...atom_site / chem_comp_atom / chem_comp_bond / database_2 / struct_ref
Item: _atom_site.auth_atom_id / _atom_site.label_atom_id ..._atom_site.auth_atom_id / _atom_site.label_atom_id / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref.pdbx_seq_one_letter_code
Revision 2.1Nov 6, 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
Revision 2.2Nov 13, 2024Group: Data collection / Structure summary / Category: em_admin / em_entity_assembly_molwt / Item: _em_admin.last_update / _em_entity_assembly_molwt.id

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Structure visualization

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Assembly

Deposited unit
A: Gamma-aminobutyric acid receptor subunit beta-2
B: Gamma-aminobutyric acid receptor subunit alpha-1
C: Gamma-aminobutyric acid receptor subunit beta-2
D: Gamma-aminobutyric acid receptor subunit alpha-1
E: Gamma-aminobutyric acid receptor subunit gamma-2
I: Kappa Fab Light Chain
J: IgG2b Fab Heavy Chain
L: Kappa Fab Light Chain
K: IgG2b Fab Heavy Chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)364,70320
Polymers360,0509
Non-polymers4,65311
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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

#1: Protein Gamma-aminobutyric acid receptor subunit beta-2 / GABA(A) receptor subunit beta-2


Mass: 41810.086 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GABRB2 / Plasmid: pEZT-BM / Cell line (production host): HEK293S / Production host: Homo sapiens (human) / Variant (production host): GnTI- / References: UniProt: P47870
#2: Protein Gamma-aminobutyric acid receptor subunit alpha-1 / GABA(A) receptor subunit alpha-1


Mass: 41061.211 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GABRA1 / Plasmid: pEZT-BM / Cell line (production host): HEK293S / Production host: Homo sapiens (human) / Variant (production host): GnTI- / References: UniProt: P14867
#3: Protein Gamma-aminobutyric acid receptor subunit gamma-2 / GABA(A) receptor subunit gamma-2


Mass: 47673.109 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GABRG2 / Plasmid: pEZT-BM / Cell line (production host): HEK293S / Production host: Homo sapiens (human) / Variant (production host): GnTI- / References: UniProt: P18507

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Antibody , 2 types, 4 molecules ILJK

#4: Antibody Kappa Fab Light Chain


Mass: 23505.943 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse)
#5: Antibody IgG2b Fab Heavy Chain


Mass: 49811.043 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse)

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Sugars , 4 types, 7 molecules

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


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}}LINUCSPDB-CARE
#7: Polysaccharide alpha-D-mannopyranose-(1-2)-alpha-D-mannopyranose-(1-2)-alpha-D-mannopyranose-(1-3)-[alpha-D- ...alpha-D-mannopyranose-(1-2)-alpha-D-mannopyranose-(1-2)-alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-2)-alpha-D-mannopyranose-(1-6)-[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: 1721.527 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-2DManpa1-2DManpa1-3[DManpa1-2DManpa1-6[DManpa1-3]DManpa1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/3,10,9/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3-3-3-3-3-3/a4-b1_b4-c1_c3-d1_c6-g1_d2-e1_e2-f1_g3-h1_g6-i1_i2-j1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{[(2+1)][a-D-Manp]{[(2+1)][a-D-Manp]{}}}[(6+1)][a-D-Manp]{[(3+1)][a-D-Manp]{}[(6+1)][a-D-Manp]{[(2+1)][a-D-Manp]{}}}}}}LINUCSPDB-CARE
#8: 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.: 1
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
#9: 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.: 3
Source method: isolated from a genetically manipulated source
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 , 2 types, 4 molecules

#10: Chemical ChemComp-ABU / GAMMA-AMINO-BUTANOIC ACID / GAMMA(AMINO)-BUTYRIC ACID


Mass: 103.120 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C4H9NO2
#11: Chemical ChemComp-UQA / 5-ethyl-5-phenylpyrimidine-2,4,6(1H,3H,5H)-trione / Phenobarbital


Mass: 232.235 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C12H12N2O3 / Feature type: SUBJECT OF INVESTIGATION / Comment: medication*YM

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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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Human GABA-A receptor alpha1-beta2-gamma2 subtype in complex with GABA and phenobarbitalCOMPLEXFab fragments generated by proteolytic cleavage of IgG antibody#1-#50MULTIPLE SOURCES
2Human GABA-A receptor alpha1-beta2-gamma2 subtypeCOMPLEX#1-#31RECOMBINANT
3IgG2b/kappa antibodyCOMPLEXFab fragment generated by proteolytic cleavage of IgG antibody#4-#51NATURAL
Molecular weight
IDEntity assembly-IDExperimental value
11NO
21NO
31NO
43
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Mus musculus (house mouse)10090
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisC4H11NO31
2150 mMSodium chlorideNaCl1
SpecimenConc.: 8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: 3.5 second blot

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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
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 69.59 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 6682

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
EM software
IDNameCategoryDetails
1SPHIREparticle selectioncrYOLO
2SerialEMimage acquisition
4CTFFINDCTF correction
7Cootmodel fitting
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
11RELIONclassification
12RELION3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1076196
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 145958 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
RefinementHighest resolution: 3.3 Å
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00817835
ELECTRON MICROSCOPYf_angle_d1.13224256
ELECTRON MICROSCOPYf_dihedral_angle_d16.76110513
ELECTRON MICROSCOPYf_chiral_restr0.0712785
ELECTRON MICROSCOPYf_plane_restr0.0082992

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