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- EMDB-8923: Cryo-EM structure of the benzodiazepine-sensitive alpha1-beta1-ga... -

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Basic information

Entry
Database: EMDB / ID: EMD-8923
TitleCryo-EM structure of the benzodiazepine-sensitive alpha1-beta1-gamma2 heterotrimeric GABAA receptor in complex with GABA
Map dataBenzodiazepine-sensitive alpha1-beta1-gamma2 heterotrimeric GABAA receptor in complex with GABA
Sample
  • Complex: Triheteromeric alpha1-beta1-gamma2 GABAA receptor
    • Protein or peptide: Gamma-aminobutyric acid receptor subunit gamma-2
    • Protein or peptide: Gamma-aminobutyric acid receptor subunit alpha-1,Gamma-aminobutyric acid receptor subunit alpha-1
    • Protein or peptide: Gamma-aminobutyric acid receptor subunit beta-1,Gamma-aminobutyric acid receptor subunit beta-1
  • Ligand: GAMMA-AMINO-BUTANOIC ACID
KeywordsNeurotransmission / GABA Receptors / GABA / Cys Loop Receptors / Ion Channel / MEMBRANE PROTEIN
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 / cellular response to histamine ...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 / cellular response to histamine / GABA-gated chloride ion channel activity / 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 / response to toxic substance / cytoplasmic vesicle membrane / 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 / 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-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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsPhulera S / Zhu H
Funding support United States, 2 items
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, 2018-
Header (metadata) releaseAug 1, 2018-
Map releaseAug 8, 2018-
UpdateNov 15, 2023-
Current statusNov 15, 2023Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 7.68
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 7.68
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6dw1
  • Surface level: 7.68
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6dw1
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_8923.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBenzodiazepine-sensitive alpha1-beta1-gamma2 heterotrimeric GABAA receptor in complex with GABA
Voxel sizeX=Y=Z: 0.649 Å
Density
Contour LevelBy AUTHOR: 7.68 / Movie #1: 7.68
Minimum - Maximum-15.883044999999999 - 32.438450000000003
Average (Standard dev.)-0.000000000002562 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 311.52 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.6490.6490.649
M x/y/z480480480
origin x/y/z0.0000.0000.000
length x/y/z311.520311.520311.520
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ480480480
MAP C/R/S321
start NC/NR/NS000
NC/NR/NS480480480
D min/max/mean-15.88332.438-0.000

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Supplemental data

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Sample components

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Entire : Triheteromeric alpha1-beta1-gamma2 GABAA receptor

EntireName: Triheteromeric alpha1-beta1-gamma2 GABAA receptor
Components
  • Complex: Triheteromeric alpha1-beta1-gamma2 GABAA receptor
    • Protein or peptide: Gamma-aminobutyric acid receptor subunit gamma-2
    • Protein or peptide: Gamma-aminobutyric acid receptor subunit alpha-1,Gamma-aminobutyric acid receptor subunit alpha-1
    • Protein or peptide: Gamma-aminobutyric acid receptor subunit beta-1,Gamma-aminobutyric acid receptor subunit beta-1
  • Ligand: GAMMA-AMINO-BUTANOIC ACID

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Supramolecule #1: Triheteromeric alpha1-beta1-gamma2 GABAA receptor

SupramoleculeName: Triheteromeric alpha1-beta1-gamma2 GABAA receptor / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3 / Details: In complex with alpha1 specific Fab
Source (natural)Organism: Rattus norvegicus (Norway rat)

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Macromolecule #1: Gamma-aminobutyric acid receptor subunit gamma-2

MacromoleculeName: Gamma-aminobutyric acid receptor subunit gamma-2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 56.894949 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MSSPNTWSTG STVYSPVFSQ KMTLWILLLL SLYPGFTSQK SDDDYEDYAS NKTWVLTPKV PEGDVTVILN NLLEGYDNKL RPDIGVKPT LIHTDMYVNS IGPVNAINME YTIDIFFAQT WYDRRLKFNS TIKVLRLNSN MVGKIWIPDT FFRNSKKADA H WITTPNRM ...String:
MSSPNTWSTG STVYSPVFSQ KMTLWILLLL SLYPGFTSQK SDDDYEDYAS NKTWVLTPKV PEGDVTVILN NLLEGYDNKL RPDIGVKPT LIHTDMYVNS IGPVNAINME YTIDIFFAQT WYDRRLKFNS TIKVLRLNSN MVGKIWIPDT FFRNSKKADA H WITTPNRM LRIWNDGRVL YTLRLTIDAE CQLQLHNFPM DEHSCPLEFS SYGYPREEIV YQWKRSSVEV GDTRSWRLYQ FS FVGLRNT TEVVKTTSGD YVVMSVYFDL SRRMGYFTIQ TYIPCTLIVV LSWVSFWINK DAVPARTSLG ITTVLTMTTL STI ARKSLP KVSYVTAMDL FVSVCFIFVF SALVEYGTLH YFVSNRKPSK DKDKKKKNPA PTIDIRPRSA TIQMNNATHL QERD EEYGY ECLDGKDCAS FFCCFEDCRT GAWRHGRIHI RIAKMDSYAR IFFPTAFCLF NLVYWVSYLY LLVPRGSRHH HHHHH HTET SQVAPA

UniProtKB: Gamma-aminobutyric acid receptor subunit gamma-2

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Macromolecule #2: Gamma-aminobutyric acid receptor subunit alpha-1,Gamma-aminobutyr...

MacromoleculeName: Gamma-aminobutyric acid receptor subunit alpha-1,Gamma-aminobutyric acid receptor subunit alpha-1
type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 46.267105 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MKKSRGLSDY LWAWTLILST LSGRSYGQPS QDELKDNTTV FTRILDRLLD GYDNRLRPGL GERVTEVKTD IFVTSFGPVS DHDMEYTID VFFRQSWKDE RLKFKGPMTV LRLNNLMASK IWTPDTFFHN GKKSVAHNMT MPNKLLRITE DGTLLYTMRL T VRAECPMH ...String:
MKKSRGLSDY LWAWTLILST LSGRSYGQPS QDELKDNTTV FTRILDRLLD GYDNRLRPGL GERVTEVKTD IFVTSFGPVS DHDMEYTID VFFRQSWKDE RLKFKGPMTV LRLNNLMASK IWTPDTFFHN GKKSVAHNMT MPNKLLRITE DGTLLYTMRL T VRAECPMH LEDFPMDAHA CPLKFGSYAY TRAEVVYEWT REPARSVVVA EDGSRLNQYD LLGQTVDSGI VQSSTGEYVV MT THFHLKR KIGYFVIQTY LPCIMTVILS QVSFWLNRES VPARTVFGVT TVLTMTTLSI SARNSLPKVA YATAMDWFIA VCY AFVFSA LIEFATVNYF TKRGTKKTFN SVSKIDRLSR IAFPLLFGIF NLVYWATYLN REPQLKAPTP HQLVPRGSHH HHHH HH

UniProtKB: Gamma-aminobutyric acid receptor subunit alpha-1, Gamma-aminobutyric acid receptor subunit alpha-1

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Macromolecule #3: Gamma-aminobutyric acid receptor subunit beta-1,Gamma-aminobutyri...

MacromoleculeName: Gamma-aminobutyric acid receptor subunit beta-1,Gamma-aminobutyric acid receptor subunit beta-1
type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 44.333012 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MWTVQNRESL GLLSFPVMVA MVCCAHSSNE PSNMSYVKET VDRLLKGYDI RLRPDFGGPP VDVGMRIDVA SIDMVSEVNM DYTLTMYFQ QSWKDKRLSY SGIPLNLTLD NRVADQLWVP DTYFLNDKKS FVHGVTVKNR MIRLHPDGTV LYGLRITTTA A CMMDLRRY ...String:
MWTVQNRESL GLLSFPVMVA MVCCAHSSNE PSNMSYVKET VDRLLKGYDI RLRPDFGGPP VDVGMRIDVA SIDMVSEVNM DYTLTMYFQ QSWKDKRLSY SGIPLNLTLD NRVADQLWVP DTYFLNDKKS FVHGVTVKNR MIRLHPDGTV LYGLRITTTA A CMMDLRRY PLDEQNCTLE IESYGYTTDD IEFYWNGGEG AVTGVNKIEL PQFSIVDYKM VSKKVEFTTG AYPRLSLSFR LK RNIGYFI LQTYMPSTLI TILSWVSFWI NYDASAARVA LGITTVLTMT TISTHLRETL PKIPYVKAID IYLMGCFVFV FLA LLEYAF VNYIFFGGTI PDLTDVNSID KWSRMFFPIT FSLFNVVYWL YYVHLVPRGS HHHHHHHH

UniProtKB: Gamma-aminobutyric acid receptor subunit beta-1, Gamma-aminobutyric acid receptor subunit beta-1

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Macromolecule #8: GAMMA-AMINO-BUTANOIC ACID

MacromoleculeName: GAMMA-AMINO-BUTANOIC ACID / type: ligand / ID: 8 / Number of copies: 3 / Formula: ABU
Molecular weightTheoretical: 103.12 Da
Chemical component information

ChemComp-ABU:
GAMMA-AMINO-BUTANOIC ACID / neurotransmitter, inhibitor*YM / Γ-Aminobutyric acid

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.15 mg/mL
BufferpH: 7.3
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
200.0 mMNaClSodium chloridesodium chloride
1.0 mMC24H46O11Dodecyl Maltoside
1.5 mMC4H9NO2gamma-Aminobutyric acid
GridSupport film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Details: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: -2.5 µm / Nominal defocus min: -1.2 µm / Nominal magnification: 120000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 1391 / Average exposure time: 40.0 sec. / Average electron dose: 37.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: cryoSPARC was used to generate the initial model
Initial angle assignmentType: OTHER / Software - Name: cryoSPARC / Details: Stochastic Gradient Descent
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 68499

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: 0.74
Output model

PDB-6dw1:
Cryo-EM structure of the benzodiazepine-sensitive alpha1beta1gamma2S tri-heteromeric GABAA receptor in complex with GABA (ECD map)

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