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- PDB-6x3v: Human GABAA receptor alpha1-beta2-gamma2 subtype in complex with ... -

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Basic information

Entry
Database: PDB / ID: 6x3v
TitleHuman GABAA receptor alpha1-beta2-gamma2 subtype in complex with GABA plus etomidate
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 / 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 ...benzodiazepine receptor activity / GABA receptor complex / 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 / postsynaptic specialization membrane / gamma-aminobutyric acid signaling pathway / synaptic transmission, GABAergic / chloride channel activity / adult behavior / Signaling by ERBB4 / cochlea development / chloride channel complex / dendrite membrane / cytoplasmic vesicle membrane / chloride transmembrane transport / post-embryonic development / transmitter-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / GABA-ergic synapse / chemical synaptic transmission / dendritic spine / postsynaptic membrane / postsynapse / axon / 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, 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. ...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, 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
Similarity search - Domain/homology
GAMMA-AMINO-BUTANOIC ACID / Etomidate / 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: 3.5 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_DOI / _citation.pdbx_database_id_PubMed ..._citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Sep 23, 2020Group: 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 / 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 3.0Jan 31, 2024Group: Atomic model / Data collection ...Atomic model / Data collection / Derived calculations / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / entity / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / struct_conn
Item: _atom_site.auth_seq_id / _chem_comp.name ..._atom_site.auth_seq_id / _chem_comp.name / _entity.pdbx_description / _pdbx_entity_nonpoly.name / _pdbx_nonpoly_scheme.pdb_seq_num / _struct_conn.ptnr2_auth_seq_id
Revision 3.1Nov 13, 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

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  • Deposited structure unit
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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)365,09220
Polymers360,0509
Non-polymers5,04311
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


Mass: 41810.086 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Plasmid: pEZT-BM / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / Variant (production host): GNTI- / References: UniProt: P47870
#2: Protein Gamma-aminobutyric acid 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) / 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


Mass: 47673.109 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / 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.: 3
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-Glycam 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
[]{[(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]{[(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.: 2
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 / Feature type: SUBJECT OF INVESTIGATION
#11: Chemical ChemComp-V8D / Etomidate


Mass: 244.289 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C14H16N2O2 / Feature type: SUBJECT OF INVESTIGATION

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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 etomidateCOMPLEXFab fragments generated by proteolytic cleavage of IgG antibody#1-#50MULTIPLE SOURCES
2Human GABA-A receptor alpha1-beta2-gamma2 subtypeCOMPLEX#1-#31RECOMBINANT
Molecular weight
IDEntity assembly-IDExperimental value
11NO
21NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Homo sapiens (human)9606
22Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
11Homo sapiens (human)9606
22Homo sapiens (human)9606
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisC4H11NO31
2150 mMSodium chlorideNaCl1
SpecimenConc.: 8.5 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: 66.07 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 9013

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
EM software
IDNameCategory
1RELIONparticle selection
2SerialEMimage acquisition
4GctfCTF correction
7Cootmodel fitting
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
11RELIONclassification
12RELION3D reconstruction
19PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1972936
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 124310 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00517862
ELECTRON MICROSCOPYf_angle_d0.55224288
ELECTRON MICROSCOPYf_dihedral_angle_d23.5442546
ELECTRON MICROSCOPYf_chiral_restr0.0442809
ELECTRON MICROSCOPYf_plane_restr0.0042988

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