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- PDB-6dw1: Cryo-EM structure of the benzodiazepine-sensitive alpha1beta1gamm... -

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Basic information

Entry
Database: PDB / ID: 6dw1
TitleCryo-EM structure of the benzodiazepine-sensitive alpha1beta1gamma2S tri-heteromeric GABAA receptor in complex with GABA (ECD map)
Components(Gamma-aminobutyric acid receptor subunit ...) x 3
KeywordsMEMBRANE PROTEIN / Neurotransmission / GABA Receptors / GABA / Cys Loop Receptors / Ion Channel
Function / homology
Function and homology information


GABA receptor activation / diazepam binding / G protein-coupled neurotransmitter receptor activity involved in regulation of presynaptic membrane potential / monoatomic anion channel activity / chemical synaptic transmission, postsynaptic / inhibitory synapse / GABA receptor complex / organic cyclic compound binding / inhibitory extracellular ligand-gated monoatomic ion channel activity / GABA-gated chloride ion channel activity ...GABA receptor activation / diazepam binding / G protein-coupled neurotransmitter receptor activity involved in regulation of presynaptic membrane potential / monoatomic anion channel activity / chemical synaptic transmission, postsynaptic / inhibitory synapse / GABA receptor complex / organic cyclic compound binding / inhibitory extracellular ligand-gated monoatomic ion channel activity / GABA-gated chloride ion channel activity / cellular response to histamine / inhibitory synapse assembly / GABA-A receptor activity / GABA-A receptor complex / GABA receptor binding / neurotransmitter receptor activity / postsynaptic specialization membrane / gamma-aminobutyric acid signaling pathway / synaptic transmission, GABAergic / chloride transport / ovulation cycle / adult behavior / chloride channel activity / central nervous system neuron development / ligand-gated monoatomic ion channel activity / chloride channel complex / GABA-ergic synapse / regulation of postsynaptic membrane potential / monoatomic ion transport / presynaptic active zone membrane / chloride transmembrane transport / dendrite membrane / ligand-gated monoatomic ion channel activity involved in regulation of presynaptic membrane potential / monoatomic ion transmembrane transport / post-embryonic development / transmitter-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / response to progesterone / Schaffer collateral - CA1 synapse / cytoplasmic vesicle membrane / response to toxic substance / nuclear envelope / chemical synaptic transmission / postsynapse / receptor complex / neuron projection / axon / dendrite / glutamatergic synapse / synapse / 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 / Acetylcholine Binding Protein; Chain: A, / Neurotransmitter-gated ion-channel ligand-binding domain / Gamma-aminobutyric acid A receptor/Glycine receptor alpha ...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 / Acetylcholine Binding Protein; Chain: A, / Neurotransmitter-gated ion-channel ligand-binding domain / 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 / Distorted Sandwich / Mainly Beta
Similarity search - Domain/homology
GAMMA-AMINO-BUTANOIC ACID / Gamma-aminobutyric acid receptor subunit beta-1 / Gamma-aminobutyric acid receptor subunit gamma-2 / Gamma-aminobutyric acid receptor subunit alpha-1
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsPhulera, S. / Zhu, H. / Yu, J. / Yoshioka, C. / Gouaux, E.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM100400 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Elife / Year: 2018
Title: Cryo-EM structure of the benzodiazepine-sensitive α1β1γ2S tri-heteromeric GABA receptor in complex with GABA.
Authors: Swastik Phulera / Hongtao Zhu / Jie Yu / Derek P Claxton / Nate Yoder / Craig Yoshioka / Eric Gouaux /
Abstract: Fast inhibitory neurotransmission in the mammalian nervous system is largely mediated by GABA receptors, chloride-selective members of the superfamily of pentameric Cys-loop receptors. Native GABA ...Fast inhibitory neurotransmission in the mammalian nervous system is largely mediated by GABA receptors, chloride-selective members of the superfamily of pentameric Cys-loop receptors. Native GABA receptors are heteromeric assemblies sensitive to many important drugs, from sedatives to anesthetics and anticonvulsant agents, with mutant forms of GABA receptors implicated in multiple neurological diseases. Despite the profound importance of heteromeric GABA receptors in neuroscience and medicine, they have proven recalcitrant to structure determination. Here we present the structure of a tri-heteromeric α1β1γ2S GABA receptor in complex with GABA, determined by single particle cryo-EM at 3.1-3.8 Å resolution, elucidating molecular principles of receptor assembly and agonist binding. Remarkable N-linked glycosylation on the α1 subunit occludes the extracellular vestibule of the ion channel and is poised to modulate receptor assembly and perhaps ion channel gating. Our work provides a pathway to structural studies of heteromeric GABA receptors and a framework for rational design of novel therapeutic agents.
History
DepositionJun 26, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 8, 2018Provider: repository / Type: Initial release
Revision 1.1Nov 20, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 2.0Jul 29, 2020Group: Atomic model / Data collection ...Atomic model / Data collection / Derived calculations / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / em_entity_assembly / entity / pdbx_branch_scheme / pdbx_chem_comp_identifier / pdbx_entity_branch / pdbx_entity_branch_descriptor / pdbx_entity_branch_link / pdbx_entity_branch_list / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_struct_assembly_gen / struct_asym / struct_conn / struct_site / struct_site_gen
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.label_asym_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.label_entity_id / _atom_site.type_symbol / _chem_comp.name / _chem_comp.type / _em_entity_assembly.entity_id_list / _pdbx_struct_assembly_gen.asym_id_list / _struct_conn.pdbx_role / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 3.0Nov 15, 2023Group: Atomic model / Data collection ...Atomic model / Data collection / Database references / Derived calculations / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / chem_comp_atom / chem_comp_bond / database_2 / struct_conn
Item: _atom_site.auth_atom_id / _atom_site.label_atom_id ..._atom_site.auth_atom_id / _atom_site.label_atom_id / _chem_comp.pdbx_synonyms / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_leaving_atom_flag

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Structure visualization

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)242,41014
Polymers238,0955
Non-polymers4,3159
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area20450 Å2
ΔGint8 kcal/mol
Surface area44730 Å2
MethodPISA

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Components

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

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


Mass: 56894.949 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Gabrg2 / Production host: Homo sapiens (human) / References: UniProt: P18508
#2: Protein Gamma-aminobutyric acid receptor subunit alpha-1,Gamma-aminobutyric acid receptor subunit alpha-1 / GABA(A) receptor subunit alpha-1


Mass: 46267.105 Da / Num. of mol.: 2 / Fragment: residues 39-334
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Gabra1, Gabra-1 / Production host: Homo sapiens (human) / References: UniProt: P62813
#3: Protein Gamma-aminobutyric acid receptor subunit beta-1,Gamma-aminobutyric acid receptor subunit beta-1 / GABA(A) receptor subunit beta-1


Mass: 44333.012 Da / Num. of mol.: 2 / Fragment: residues 33-474
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Gabrb1, Gabrb-1 / Production host: Homo sapiens (human) / References: UniProt: P15431

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Sugars , 4 types, 6 molecules

#4: Polysaccharide alpha-D-mannopyranose-(1-3)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1- ...alpha-D-mannopyranose-(1-3)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 748.682 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/3,4,3/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3/a4-b1_b4-c1_c3-d1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}}}}}LINUCSPDB-CARE
#5: Polysaccharide alpha-D-mannopyranose-(1-2)-alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-3)-alpha-D- ...alpha-D-mannopyranose-(1-2)-alpha-D-mannopyranose-(1-3)-[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: 1235.105 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-2DManpa1-3[DManpa1-3DManpa1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/3,7,6/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3-3-3/a4-b1_b4-c1_c3-d1_c6-f1_d2-e1_f3-g1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{[(2+1)][a-D-Manp]{}}[(6+1)][a-D-Manp]{[(3+1)][a-D-Manp]{}}}}}}LINUCSPDB-CARE
#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 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.: 2
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

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Non-polymers , 1 types, 3 molecules

#8: Chemical ChemComp-ABU / GAMMA-AMINO-BUTANOIC ACID / GAMMA(AMINO)-BUTYRIC ACID / Γ-Aminobutyric acid


Mass: 103.120 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C4H9NO2 / Comment: neurotransmitter, inhibitor*YM

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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: Triheteromeric alpha1-beta1-gamma2 GABAA receptor / Type: COMPLEX / Details: In complex with alpha1 specific Fab / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightUnits: KILODALTONS/NANOMETER / Experimental value: NO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Source (recombinant)Organism: Homo sapiens (human) / Cell: TSA201
Buffer solutionpH: 7.3
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2200 mMsodium chlorideNaClSodium chloride1
31 mMDodecyl MaltosideC24H46O111
41.5 mMgamma-Aminobutyric acidC4H9NO21
SpecimenConc.: 0.15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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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: 120000 X / Nominal defocus max: -2500 nm / Nominal defocus min: -1200 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 40 sec. / Electron dose: 37 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1391

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Processing

EM software
IDNameVersionCategory
1DoG Pickerparticle selection
2EPUimage acquisition
4Gctf1.06CTF correction
5RELION2.1CTF correction
8Coot0.8.6.1model fitting
10PHENIX1.13model refinement
11cryoSPARCinitial Euler assignment
12RELION2.1final Euler assignment
14RELION2.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 68499 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: 0.74

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