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- EMDB-29727: Native GABA-A receptor from the mouse brain, alpha1-beta2-gamma2 ... -

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Entry
Database: EMDB / ID: EMD-29727
TitleNative GABA-A receptor from the mouse brain, alpha1-beta2-gamma2 subtype, in complex with GABA, Zolpidem, and endogenous neurosteroids
Map dataNative GABA receptor in complex with 8E3-Fab, Zolpidem, and endogenous neurosteroids
Sample
  • Complex: Native GABAA Receptor isolated from mouse brain containing two alpha1 subunits, in complex with 8E3-Fab, GABA, Zolpidem, and endogenous neurosteroid
    • Protein or peptide: x 2 types
  • Protein or peptide: x 3 types
  • Ligand: x 8 types
Keywordsligand-gated ion channel / cys-loop receptor / neurotransmitter receptor / MEMBRANE PROTEIN
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 / postsynaptic specialization membrane / neurotransmitter receptor activity / synaptic transmission, GABAergic / gamma-aminobutyric acid signaling pathway / chloride channel activity / adult behavior / 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 / cytoplasmic vesicle / chemical synaptic transmission / postsynapse / neuron projection / axon / synapse / glutamatergic 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 gamma-2 / Gamma-aminobutyric acid receptor subunit alpha-1 / Gamma-aminobutyric acid receptor subunit beta-2
Similarity search - Component
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.67 Å
AuthorsSun C / Gouaux E
Funding support United States, 2 items
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
CitationJournal: 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.
History
DepositionFeb 10, 2023-
Header (metadata) releaseSep 20, 2023-
Map releaseSep 20, 2023-
UpdateNov 15, 2023-
Current statusNov 15, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29727.png

Downloads & links

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Map

FileDownload / File: emd_29727.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNative GABA receptor in complex with 8E3-Fab, Zolpidem, and endogenous neurosteroids
Voxel sizeX=Y=Z: 0.826 Å
Density
Contour LevelBy AUTHOR: 0.11
Minimum - Maximum-0.29468456 - 0.6234526
Average (Standard dev.)0.0021136494 (±0.019460866)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 297.36 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_29727_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_29727_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_29727_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Native GABAA Receptor isolated from mouse brain containing two al...

EntireName: Native GABAA Receptor isolated from mouse brain containing two alpha1 subunits, in complex with 8E3-Fab, GABA, Zolpidem, and endogenous neurosteroid
Components
  • Complex: Native GABAA Receptor isolated from mouse brain containing two alpha1 subunits, in complex with 8E3-Fab, GABA, Zolpidem, and endogenous neurosteroid
    • Protein or peptide: Heavy Chain of 8E3 Fab
    • Protein or peptide: Light Chain of 8E3 Fab
  • Protein or peptide: Gamma-aminobutyric acid receptor subunit alpha-1
  • Protein or peptide: Gamma-aminobutyric acid receptor subunit beta-2
  • Protein or peptide: Gamma-aminobutyric acid receptor subunit gamma-2
  • Ligand: allopregnanolone
  • Ligand: [(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
  • Ligand: DODECANE
  • Ligand: N-OCTANEOctane
  • Ligand: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate
  • Ligand: GAMMA-AMINO-BUTANOIC ACID
  • Ligand: Zolpidem
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Native GABAA Receptor isolated from mouse brain containing two al...

SupramoleculeName: Native GABAA Receptor isolated from mouse brain containing two alpha1 subunits, in complex with 8E3-Fab, GABA, Zolpidem, and endogenous neurosteroid
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #4-#5
Source (natural)Organism: Mus musculus (house mouse) / Strain: C57BL/6 / Organ: Brain
Molecular weightTheoretical: 350 KDa

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Macromolecule #1: Gamma-aminobutyric acid receptor subunit alpha-1

MacromoleculeName: Gamma-aminobutyric acid receptor subunit alpha-1 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse) / Strain: C57BL/6
Molecular weightTheoretical: 51.818516 KDa
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 TKRGYAWDGK SVVPEKPKKV KDPLIKKNNT YAPTATSYTP NLARGDPGLA TIAKSATIEP KEVK PETKP PEPKKTFNSV SKIDRLSRIA FPLLFGIFNL VYWATYLNRE PQLKAPTPHQ

UniProtKB: Gamma-aminobutyric acid receptor subunit alpha-1

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Macromolecule #2: Gamma-aminobutyric acid receptor subunit beta-2

MacromoleculeName: Gamma-aminobutyric acid receptor subunit beta-2 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse) / Strain: C57BL/6
Molecular weightTheoretical: 59.265312 KDa
SequenceString: MWRVRKRGYF GIWSFPLIIA AVCAQSVNDP SNMSLVKETV DRLLKGYDIR LRPDFGGPPV AVGMNIDIAS IDMVSEVNMD YTLTMYFQQ AWRDKRLSYN VIPLNLTLDN RVADQLWVPD TYFLNDKKSF VHGVTVKNRM IRLHPDGTVL YGLRITTTAA C MMDLRRYP ...String:
MWRVRKRGYF GIWSFPLIIA AVCAQSVNDP SNMSLVKETV DRLLKGYDIR LRPDFGGPPV AVGMNIDIAS IDMVSEVNMD YTLTMYFQQ AWRDKRLSYN VIPLNLTLDN RVADQLWVPD TYFLNDKKSF VHGVTVKNRM IRLHPDGTVL YGLRITTTAA C MMDLRRYP LDEQNCTLEI ESYGYTTDDI EFYWRGDDNA VTGVTKIELP QFSIVDYKLI TKKVVFSTGS YPRLSLSFKL KR NIGYFIL QTYMPSILIT ILSWVSFWIN YDASAARVAL GITTVLTMTT INTHLRETLP KIPYVKAIDM YLMGCFVFVF MAL LEYALV NYIFFGRGPQ RQKKAAEKAA NANNEKMRLD VNKMFYKDIK QNGTQYRSLW DPTGDLSPTR RTTNYDFSLY TMDP HENIL LSTLEIKNEM ATSEAVMGLG DPRSTMLAYD ASSIQYRKAG LPRHSFGRNA LERHVAQKKS RLRRRASQLK ITIPD LTDV NAIDRWSRIF FPVVFSFFNI VYWLYYVN

UniProtKB: Gamma-aminobutyric acid receptor subunit beta-2

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Macromolecule #3: Gamma-aminobutyric acid receptor subunit gamma-2

MacromoleculeName: Gamma-aminobutyric acid receptor subunit gamma-2 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse) / Strain: C57BL/6
Molecular weightTheoretical: 55.160281 KDa
SequenceString: MSSPNTWSIG SSVYSPVFSQ KMTLWILLLL SLYPGFTSQK SDDDYEDYAS NKTWVLTPKV PEGDVTVILN NLLEGYDNKL RPDIGVKPT LIHTDMYVNS IGPVNAINME YTIDIFFAQT WYDRRLKFNS TIKVLRLNSN MVGKIWIPDT FFRNSKKADA H WITTPNRM ...String:
MSSPNTWSIG SSVYSPVFSQ 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 DKDKKKKNPL LRMFSFKAPT IDIRPRSATI QMNN ATHLQ ERDEEYGYEC LDGKDCASFF CCFEDCRTGA WRHGRIHIRI AKMDSYARIF FPTAFCLFNL VYWVSYLYL

UniProtKB: Gamma-aminobutyric acid receptor subunit gamma-2

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Macromolecule #4: Heavy Chain of 8E3 Fab

MacromoleculeName: Heavy Chain of 8E3 Fab / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 24.159895 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: EIQLQQSGPE LVKPGTSVKV SCKASGYSFT DYNMYWVKQS HGKSLEWIGY IDPYNADTTY NREFKGKATL TVDKSSSTAF MHLNSLTSE DSAVYYCARK RNNFYFDYWG QGTPLTVSSA KTTPPSVYPL APGCGDTTGS SVTLGCLVKG YFPESVTVTW N SGSLSSSV ...String:
EIQLQQSGPE LVKPGTSVKV SCKASGYSFT DYNMYWVKQS HGKSLEWIGY IDPYNADTTY NREFKGKATL TVDKSSSTAF MHLNSLTSE DSAVYYCARK RNNFYFDYWG QGTPLTVSSA KTTPPSVYPL APGCGDTTGS SVTLGCLVKG YFPESVTVTW N SGSLSSSV HTFPALLQSG LYTMSSSVTV PSSTWPSQTV TCSVAHPASS TTVDKKSAAL EVLFQ

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Macromolecule #5: Light Chain of 8E3 Fab

MacromoleculeName: Light Chain of 8E3 Fab / type: protein_or_peptide / ID: 5 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 23.62408 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: YIVMTQSPKS MSMSLGERVT LSCRASEYVG SYVSWYQQKP EQSPKLLIYG ASNRYTGVPD RFAGSGSATD FTLTITSVQA EDLADYHCG QTYNYPTFGG GTKLEIKRAD AAPTVSIFPP SSEQLTSGGA SVVCFLNNFY PKDINVKWKI DGSERQNGVL N SWTDQDSK ...String:
YIVMTQSPKS MSMSLGERVT LSCRASEYVG SYVSWYQQKP EQSPKLLIYG ASNRYTGVPD RFAGSGSATD FTLTITSVQA EDLADYHCG QTYNYPTFGG GTKLEIKRAD AAPTVSIFPP SSEQLTSGGA SVVCFLNNFY PKDINVKWKI DGSERQNGVL N SWTDQDSK DSTYSMSSTL TLTKDEYERH NSYTCEATHK TSTSPIVKSF NRNEC

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Macromolecule #9: allopregnanolone

MacromoleculeName: allopregnanolone / type: ligand / ID: 9 / Number of copies: 2 / Formula: Y4B
Molecular weightTheoretical: 318.493 Da
Chemical component information

ChemComp-Y4B:
allopregnanolone

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Macromolecule #10: [(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(o...

MacromoleculeName: [(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
type: ligand / ID: 10 / Number of copies: 2 / Formula: PIO
Molecular weightTheoretical: 746.566 Da
Chemical component information

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

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Macromolecule #11: DODECANE

MacromoleculeName: DODECANE / type: ligand / ID: 11 / Number of copies: 17 / Formula: D12
Molecular weightTheoretical: 170.335 Da
Chemical component information

ChemComp-D12:
DODECANE / Dodecane

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Macromolecule #12: N-OCTANE

MacromoleculeName: N-OCTANE / type: ligand / ID: 12 / Number of copies: 17 / Formula: OCT
Molecular weightTheoretical: 114.229 Da
Chemical component information

ChemComp-OCT:
N-OCTANE / Octane

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Macromolecule #13: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(tri...

MacromoleculeName: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate
type: ligand / ID: 13 / Number of copies: 5 / Formula: POV
Molecular weightTheoretical: 760.076 Da
Chemical component information

ChemComp-POV:
(2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate / phospholipid*YM / POPC

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Macromolecule #14: GAMMA-AMINO-BUTANOIC ACID

MacromoleculeName: GAMMA-AMINO-BUTANOIC ACID / type: ligand / ID: 14 / Number of copies: 2 / 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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Macromolecule #15: Zolpidem

MacromoleculeName: Zolpidem / type: ligand / ID: 15 / Number of copies: 1 / Formula: R5R
Molecular weightTheoretical: 307.39 Da
Chemical component information

ChemComp-R5R:
Zolpidem / medication*YM / Zolpidem

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Macromolecule #16: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 16 / Number of copies: 1 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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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

Concentration0.1 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chlorideSodium Chloride
20.0 mMTris-HClTrisTris
GridModel: Quantifoil R2/1 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV
DetailsThis sample was monodisperse.

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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 / Nominal defocus max: 2.1 µm / Nominal defocus min: 0.8 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.67 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2) / Number images used: 124019
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-8g4n:
Native GABA-A receptor from the mouse brain, alpha1-beta2-gamma2 subtype, in complex with GABA, Zolpidem, and endogenous neurosteroids

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