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- PDB-8g5h: Native GABA-A receptor from the mouse brain, ortho-alpha1-alpha3-... -

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Entry
Database: PDB / ID: 8g5h
TitleNative GABA-A receptor from the mouse brain, ortho-alpha1-alpha3-beta2-gamma2 subtype, in complex with GABA, Zolpidem, and endogenous neurosteroid
Components
  • (Gamma-aminobutyric acid receptor subunit ...) x 4
  • Heavy Chain of 8E3 Fab
  • Light Chain of 8E3 Fab
KeywordsMEMBRANE PROTEIN / ligand-gated ion channel / cys-loop receptor / neurotransmitter receptor
Function / homology
Function and homology information


GABA receptor activation / diazepam binding / inhibitory synapse / GABA receptor complex / organic cyclic compound binding / inhibitory extracellular ligand-gated monoatomic ion channel activity / inner ear receptor cell development / GABA-gated chloride ion channel activity / cellular response to histamine / inhibitory synapse assembly ...GABA receptor activation / diazepam binding / inhibitory synapse / GABA receptor complex / organic cyclic compound binding / inhibitory extracellular ligand-gated monoatomic ion channel activity / inner ear receptor cell development / GABA-gated chloride ion channel activity / cellular response to histamine / inhibitory synapse assembly / GABA-A receptor activity / GABA-A receptor complex / innervation / neurotransmitter receptor activity / postsynaptic specialization membrane / gamma-aminobutyric acid signaling pathway / synaptic transmission, GABAergic / adult behavior / chloride channel activity / cochlea development / chloride channel complex / GABA-ergic synapse / neuron development / regulation of postsynaptic membrane potential / presynaptic active zone membrane / chloride transmembrane transport / dendrite membrane / ligand-gated monoatomic ion channel activity involved in regulation of presynaptic membrane potential / post-embryonic development / transmitter-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / cytoplasmic vesicle membrane / chemical synaptic transmission / cytoplasmic vesicle / postsynapse / neuron projection / axon / glutamatergic synapse / synapse / plasma membrane
Similarity search - Function
Gamma-aminobutyric-acid A receptor, alpha 3 subunit / 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 ...Gamma-aminobutyric-acid A receptor, alpha 3 subunit / 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 / Chem-PIO / Zolpidem / allopregnanolone / Gamma-aminobutyric acid receptor subunit gamma-2 / Gamma-aminobutyric acid receptor subunit alpha-3 / Gamma-aminobutyric acid receptor subunit alpha-1 / Gamma-aminobutyric acid receptor subunit beta-2
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.89 Å
AuthorsSun, C. / Gouaux, E.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5R01GM100400-08 United States
Howard Hughes Medical Institute (HHMI) United States
Citation
Journal: Nature / Year: 2023
Title: Cryo-EM structures reveal native GABA receptor assemblies and pharmacology.
Authors: Chang Sun / Hongtao Zhu / Sarah Clark / Eric Gouaux /
Abstract: Type A γ-aminobutyric acid receptors (GABARs) are the principal inhibitory receptors in the brain and the target of a wide range of clinical agents, including anaesthetics, sedatives, hypnotics and ...Type A γ-aminobutyric acid receptors (GABARs) are the principal inhibitory receptors in the brain and the target of a wide range of clinical agents, including anaesthetics, sedatives, hypnotics and antidepressants. However, our understanding of GABAR pharmacology has been hindered by the vast number of pentameric assemblies that can be derived from 19 different subunits and the lack of structural knowledge of clinically relevant receptors. Here, we isolate native murine GABAR assemblies containing the widely expressed α1 subunit and elucidate their structures in complex with drugs used to treat insomnia (zolpidem (ZOL) and flurazepam) and postpartum depression (the neurosteroid allopregnanolone (APG)). Using cryo-electron microscopy (cryo-EM) analysis and single-molecule photobleaching experiments, we uncover three major structural populations in the brain: the canonical α1β2γ2 receptor containing two α1 subunits, and two assemblies containing one α1 and either an α2 or α3 subunit, in which the single α1-containing receptors feature a more compact arrangement between the transmembrane and extracellular domains. Interestingly, APG is bound at the transmembrane α/β subunit interface, even when not added to the sample, revealing an important role for endogenous neurosteroids in modulating native GABARs. Together with structurally engaged lipids, neurosteroids produce global conformational changes throughout the receptor that modify the ion channel pore and the binding sites for GABA and insomnia medications. Our data reveal the major α1-containing GABAR assemblies, bound with endogenous neurosteroid, thus defining a structural landscape from which subtype-specific drugs can be developed.
#1: Journal: Acta Crystallogr D Struct Biol / Year: 2018
Title: Real-space refinement in PHENIX for cryo-EM and crystallography.
Authors: Pavel V Afonine / Billy K Poon / Randy J Read / Oleg V Sobolev / Thomas C Terwilliger / Alexandre Urzhumtsev / Paul D Adams /
Abstract: This article describes the implementation of real-space refinement in the phenix.real_space_refine program from the PHENIX suite. The use of a simplified refinement target function enables very fast ...This article describes the implementation of real-space refinement in the phenix.real_space_refine program from the PHENIX suite. The use of a simplified refinement target function enables very fast calculation, which in turn makes it possible to identify optimal data-restraint weights as part of routine refinements with little runtime cost. Refinement of atomic models against low-resolution data benefits from the inclusion of as much additional information as is available. In addition to standard restraints on covalent geometry, phenix.real_space_refine makes use of extra information such as secondary-structure and rotamer-specific restraints, as well as restraints or constraints on internal molecular symmetry. The re-refinement of 385 cryo-EM-derived models available in the Protein Data Bank at resolutions of 6 Å or better shows significant improvement of the models and of the fit of these models to the target maps.
History
DepositionFeb 13, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 20, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 4, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Oct 11, 2023Group: 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: Advisory / Atomic model / Data collection
Category: atom_site / chem_comp_atom ...atom_site / chem_comp_atom / chem_comp_bond / pdbx_validate_close_contact
Item: _atom_site.auth_atom_id / _atom_site.label_atom_id ..._atom_site.auth_atom_id / _atom_site.label_atom_id / _chem_comp_atom.atom_id / _chem_comp_bond.atom_id_1 / _chem_comp_bond.atom_id_2 / _pdbx_validate_close_contact.auth_atom_id_2

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Gamma-aminobutyric acid receptor subunit beta-2
C: Gamma-aminobutyric acid receptor subunit alpha-1
D: Gamma-aminobutyric acid receptor subunit gamma-2
E: Gamma-aminobutyric acid receptor subunit beta-2
J: Heavy Chain of 8E3 Fab
K: Light Chain of 8E3 Fab
A: Gamma-aminobutyric acid receptor subunit alpha-3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)337,66121
Polymers328,7557
Non-polymers8,90614
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Gamma-aminobutyric acid receptor subunit ... , 4 types, 5 molecules BECDA

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


Mass: 59265.312 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse) / Strain: C57BL/6 / References: UniProt: P63137
#2: Protein Gamma-aminobutyric acid receptor subunit alpha-1 / GABA(A) receptor subunit alpha-1


Mass: 51818.516 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse) / Strain: C57BL/6 / References: UniProt: P62812
#3: Protein Gamma-aminobutyric acid receptor subunit gamma-2 / GABA(A) receptor subunit gamma-2


Mass: 55160.281 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse) / Strain: C57BL/6 / References: UniProt: P22723
#6: Protein Gamma-aminobutyric acid receptor subunit alpha-3 / GABA(A) receptor subunit alpha-3


Mass: 55461.684 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse) / Strain: C57BL/6 / References: UniProt: P26049

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Antibody , 2 types, 2 molecules JK

#4: Antibody Heavy Chain of 8E3 Fab


Mass: 24159.895 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Spodoptera frugiperda (fall armyworm)
#5: Antibody Light Chain of 8E3 Fab


Mass: 23624.080 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Spodoptera frugiperda (fall armyworm)

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Sugars , 4 types, 8 molecules

#7: 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.: 5
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
#8: Polysaccharide alpha-D-mannopyranose-(1-3)-alpha-D-mannopyranose-(1-6)-[alpha-D-mannopyranose-(1-3)]beta-D- ...alpha-D-mannopyranose-(1-3)-alpha-D-mannopyranose-(1-6)-[alpha-D-mannopyranose-(1-3)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)]2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 1219.105 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3DManpa1-6[DManpa1-3]DManpb1-4DGlcpNAcb1-4[LFucpa1-6]DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/4,7,6/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5][a1221m-1a_1-5]/1-1-2-3-3-3-4/a4-b1_a6-g1_b4-c1_c3-d1_c6-e1_e3-f1WURCSPDB2Glycan 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]{[(3+1)][a-D-Manp]{}}}}[(6+1)][a-L-Fucp]{}}}LINUCSPDB-CARE
#9: 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.: 1 / Source method: obtained synthetically
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
#13: 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.: 1 / 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 , 4 types, 6 molecules

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


Mass: 103.120 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C4H9NO2 / Comment: neurotransmitter, inhibitor*YM
#11: 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
#12: Chemical ChemComp-R5R / Zolpidem / Zolpidem


Mass: 307.390 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C19H21N3O / Comment: medication*YM
#14: Chemical ChemComp-Y4B / allopregnanolone / 3beta-hydroxy-5beta,10alpha,14beta,17alpha-pregnan-20-one / Brexanolone


Mass: 318.493 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H34O2

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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: Native GABAA Receptor isolated from mouse brain containing one alpha1 subunit, in complex with 8E3-Fab, GABA, Zolpidem, and endogenous neurosteroid
Type: COMPLEX / Entity ID: #1-#6 / Source: NATURAL
Molecular weightValue: 0.35 MDa / Experimental value: NO
Source (natural)Organism: Mus musculus (house mouse) / Strain: C57BL/6 / Organ: Brain
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMSodium ChlorideNaClSodium chloride1
220 mMTrisTris-HClTris1
SpecimenConc.: 0.1 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 R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 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 defocus max: 2100 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM softwareName: cryoSPARC
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.89 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 91747 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
RefinementHighest resolution: 2.89 Å

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