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- EMDB-14069: Cryo-EM structure of human full-length extrasynaptic alpha4beta3d... -

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Basic information

Entry
Database: EMDB / ID: EMD-14069
TitleCryo-EM structure of human full-length extrasynaptic alpha4beta3delta GABA(A)R in complex with GABA, histamine and nanobody Nb25
Map data
Sample
  • Complex: Human full-length extrasynaptic alpha4beta3delta GABA(A)R in complex with GABA, histamine and nanobody Nb25
    • Protein or peptide: x 4 types
  • Ligand: x 7 types
Keywordspentameric ligand-gated ion channel / neurotransmitter receptor / GABA receptor / MEMBRANE PROTEIN
Function / homology
Function and homology information


benzodiazepine receptor activity / cellular response to histamine / GABA receptor activation / GABA-A receptor activity / GABA-gated chloride ion channel activity / GABA-A receptor complex / inhibitory synapse assembly / synaptic transmission, GABAergic / gamma-aminobutyric acid signaling pathway / postsynaptic specialization membrane ...benzodiazepine receptor activity / cellular response to histamine / GABA receptor activation / GABA-A receptor activity / GABA-gated chloride ion channel activity / GABA-A receptor complex / inhibitory synapse assembly / synaptic transmission, GABAergic / gamma-aminobutyric acid signaling pathway / postsynaptic specialization membrane / neurotransmitter receptor activity / chloride channel activity / roof of mouth development / Signaling by ERBB4 / chloride channel complex / regulation of postsynaptic membrane potential / transmembrane transporter complex / GABA-ergic synapse / chloride transmembrane transport / dendrite membrane / transmitter-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / cytoplasmic vesicle membrane / postsynaptic membrane / postsynapse / dendritic spine / neuron projection / axon / neuronal cell body / dendrite / synapse / signal transduction / identical protein binding / plasma membrane
Similarity search - Function
Gamma-aminobutyric-acid A receptor, alpha 4 subunit / Gamma-aminobutyric-acid A receptor delta 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 ...Gamma-aminobutyric-acid A receptor, alpha 4 subunit / Gamma-aminobutyric-acid A receptor delta 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 delta / Gamma-aminobutyric acid receptor subunit beta-3 / Gamma-aminobutyric acid receptor subunit alpha-4
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsSente A / Desai R
Funding support United Kingdom, 5 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MR/L009609/1 United Kingdom
Medical Research Council (MRC, United Kingdom)MC_UP_1201/15 United Kingdom
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1R01-GM135550 United Kingdom
Wellcome Trust206171/Z/17/Z United Kingdom
Wellcome Trust202905/Z/16/Z United Kingdom
CitationJournal: Nature / Year: 2022
Title: Differential assembly diversifies GABA receptor structures and signalling.
Authors: Andrija Sente / Rooma Desai / Katerina Naydenova / Tomas Malinauskas / Youssef Jounaidi / Jonas Miehling / Xiaojuan Zhou / Simonas Masiulis / Steven W Hardwick / Dimitri Y Chirgadze / Keith ...Authors: Andrija Sente / Rooma Desai / Katerina Naydenova / Tomas Malinauskas / Youssef Jounaidi / Jonas Miehling / Xiaojuan Zhou / Simonas Masiulis / Steven W Hardwick / Dimitri Y Chirgadze / Keith W Miller / A Radu Aricescu /
Abstract: Type A γ-aminobutyric acid receptors (GABARs) are pentameric ligand-gated chloride channels that mediate fast inhibitory signalling in neural circuits and can be modulated by essential medicines ...Type A γ-aminobutyric acid receptors (GABARs) are pentameric ligand-gated chloride channels that mediate fast inhibitory signalling in neural circuits and can be modulated by essential medicines including general anaesthetics and benzodiazepines. Human GABAR subunits are encoded by 19 paralogous genes that can, in theory, give rise to 495,235 receptor types. However, the principles that govern the formation of pentamers, the permutational landscape of receptors that may emerge from a subunit set and the effect that this has on GABAergic signalling remain largely unknown. Here we use cryogenic electron microscopy to determine the structures of extrasynaptic GABARs assembled from α4, β3 and δ subunits, and their counterparts incorporating γ2 instead of δ subunits. In each case, we identified two receptor subtypes with distinct stoichiometries and arrangements, all four differing from those previously observed for synaptic, α1-containing receptors. This, in turn, affects receptor responses to physiological and synthetic modulators by creating or eliminating ligand-binding sites at subunit interfaces. We provide structural and functional evidence that selected GABAR arrangements can act as coincidence detectors, simultaneously responding to two neurotransmitters: GABA and histamine. Using assembly simulations and single-cell RNA sequencing data, we calculated the upper bounds for receptor diversity in recombinant systems and in vivo. We propose that differential assembly is a pervasive mechanism for regulating the physiology and pharmacology of GABARs.
History
DepositionDec 20, 2021-
Header (metadata) releaseApr 13, 2022-
Map releaseApr 13, 2022-
UpdateNov 13, 2024-
Current statusNov 13, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14069.png

Downloads & links

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Map

FileDownload / File: emd_14069.map.gz / Format: CCP4 / Size: 93 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.9 Å/pix.
x 290 pix.
= 259.84 Å		0.9 Å/pix.
x 290 pix.
= 259.84 Å		0.9 Å/pix.
x 290 pix.
= 259.84 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.896 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.008
Minimum - Maximum-0.033546418 - 0.101337835
Average (Standard dev.)0.0002518301 (±0.0033367292)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions290290290
Spacing290290290
CellA=B=C: 259.84 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Human full-length extrasynaptic alpha4beta3delta GABA(A)R in comp...

EntireName: Human full-length extrasynaptic alpha4beta3delta GABA(A)R in complex with GABA, histamine and nanobody Nb25
Components
  • Complex: Human full-length extrasynaptic alpha4beta3delta GABA(A)R in complex with GABA, histamine and nanobody Nb25
    • Protein or peptide: Gamma-aminobutyric acid receptor subunit alpha-4
    • Protein or peptide: Gamma-aminobutyric acid receptor subunit beta-3
    • Protein or peptide: Gamma-aminobutyric acid receptor subunit delta
    • Protein or peptide: Nanobody Nb25
  • Ligand: 1,2-DIPALMITOYL-SN-GLYCERO-3-PHOSPHATE
  • Ligand: DECANE
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: GAMMA-AMINO-BUTANOIC ACID
  • Ligand: HEXADECANE
  • Ligand: HISTAMINE
  • Ligand: water

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Supramolecule #1: Human full-length extrasynaptic alpha4beta3delta GABA(A)R in comp...

SupramoleculeName: Human full-length extrasynaptic alpha4beta3delta GABA(A)R in complex with GABA, histamine and nanobody Nb25
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Gamma-aminobutyric acid receptor subunit alpha-4

MacromoleculeName: Gamma-aminobutyric acid receptor subunit alpha-4 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 61.696047 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MVSAKKVPAI ALSAGVSFAL LRFLCLAVCL NESPGQNQKE EKLCTENFTR ILDSLLDGYD NRLRPGFGGP VTEVKTDIYV TSFGPVSDV EMEYTMDVFF RQTWIDKRLK YDGPIEILRL NNMMVTKVWT PDTFFRNGKK SVSHNMTAPN KLFRIMRNGT I LYTMRLTI ...String:
MVSAKKVPAI ALSAGVSFAL LRFLCLAVCL NESPGQNQKE EKLCTENFTR ILDSLLDGYD NRLRPGFGGP VTEVKTDIYV TSFGPVSDV EMEYTMDVFF RQTWIDKRLK YDGPIEILRL NNMMVTKVWT PDTFFRNGKK SVSHNMTAPN KLFRIMRNGT I LYTMRLTI SAECPMRLVD FPMDGHACPL KFGSYAYPKS EMIYTWTKGP EKSVEVPKES SSLVQYDLIG QTVSSETIKS IT GEYIVMT VYFHLRRKMG YFMIQTYIPC IMTVILSQVS FWINKESVPA RTVFGITTVL TMTTLSISAR HSLPKVSYAT AMD WFIAVC FAFVFSALIE FAAVNYFTNI QMEKAKRKTS KPPQEVPAAP VQREKHPEAP LQNTNANLNM RKRTNALVHS ESDV GNRTE VGNHSSKSST VVQESSKGTP RSYLASSPNP FSRANAAETI SAARALPSAS PTSIRTGYMP RKASVGSAST RHVFG SRLQ RIKTTVNTIG ATGKLSATPP PSAPPPSGSG TSKIDKYARI LFPVTFGAFN MVYWVVYLSK DTMEKSESLM

UniProtKB: Gamma-aminobutyric acid receptor subunit alpha-4

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

MacromoleculeName: Gamma-aminobutyric acid receptor subunit beta-3 / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 54.180348 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MWGLAGGRLF GIFSAPVLVA VVCCAQSVND PGNMSFVKET VDKLLKGYDI RLRPDFGGPP VCVGMNIDIA SIDMVSEVNM DYTLTMYFQ QYWRDKRLAY SGIPLNLTLD NRVADQLWVP DTYFLNDKKS FVHGVTVKNR MIRLHPDGTV LYGLRITTTA A CMMDLRRY ...String:
MWGLAGGRLF GIFSAPVLVA VVCCAQSVND PGNMSFVKET VDKLLKGYDI RLRPDFGGPP VCVGMNIDIA SIDMVSEVNM DYTLTMYFQ QYWRDKRLAY SGIPLNLTLD NRVADQLWVP DTYFLNDKKS FVHGVTVKNR MIRLHPDGTV LYGLRITTTA A CMMDLRRY PLDEQNCTLE IESYGYTTDD IEFYWRGGDK AVTGVERIEL PQFSIVEHRL VSRNVVFATG AYPRLSLSFR LK RNIGYFI LQTYMPSILI TILSWVSFWI NYDASAARVA LGITTVLTMT TINTHLRETL PKIPYVKAID MYLMGCFVFV FLA LLEYAF VNYIFFGRGP QRQKKLAEKT AKAKNDRSKS ESNRVDAHGN ILLTSLEVHN EMNEVSGGIG DTRNSAISFD NSGI QYRKQ SMPREGHGRF LGDRSLPHKK THLRRRSSQL KIKIPDLTDV NAIDRWSRIV FPFTFSLFNL VYWLYYVN

UniProtKB: Gamma-aminobutyric acid receptor subunit beta-3

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Macromolecule #3: Gamma-aminobutyric acid receptor subunit delta

MacromoleculeName: Gamma-aminobutyric acid receptor subunit delta / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 52.437926 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MDAPARLLAP LLLLCAQQLR GTRAMNDIGD YVGSNLEISW LPNLDGLIAG YARNFRPGIG GPPVNVALAL EVASIDHISE ANMEYTMTV FLHQSWRDSR LSYNHTNETL GLDSRFVDKL WLPDTFIVNA KSAWFHDVTV ENKLIRLQPD GVILYSIRIT S TVACDMDL ...String:
MDAPARLLAP LLLLCAQQLR GTRAMNDIGD YVGSNLEISW LPNLDGLIAG YARNFRPGIG GPPVNVALAL EVASIDHISE ANMEYTMTV FLHQSWRDSR LSYNHTNETL GLDSRFVDKL WLPDTFIVNA KSAWFHDVTV ENKLIRLQPD GVILYSIRIT S TVACDMDL AKYPMDEQEC MLDLESYGYS SEDIVYYWSE SQEHIHGLDK LQLAQFTITS YRFTTELMNF KSAGQFPRLS LH FHLRRNR GVYIIQSYMP SVLLVAMSWV SFWISQAAVP ARVSLGITTV LTMTTLMVSA RSSLPRASAI KALDVYFWIC YVF VFAALV EYAFAHFNAD YRKKQKAKVK VSRPRAEMDV RNAIVLFSLS AAGVTQELAI SRRQRRVPGN LMGSYRSVGV ETGE TKKEG AARSGGQGGI RARLRPIDAD TIDIYARAVF PAAFAAVNVI YWAAYAMGGS GGSGGSGKTE TSQVAPA

UniProtKB: Gamma-aminobutyric acid receptor subunit delta

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Macromolecule #4: Nanobody Nb25

MacromoleculeName: Nanobody Nb25 / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 13.341741 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
QVQLVESGGG LVQGSLRLSC AASGHTFNYP IMGWFRQAPG KEREFVGAIS WSGGSTSYAD SVKDRFTISR DNAKNTVYLE MNNLKPEDT AVYYCAAKGR YSGGLYYPTN YDYWGQGTQV TV

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Macromolecule #7: 1,2-DIPALMITOYL-SN-GLYCERO-3-PHOSPHATE

MacromoleculeName: 1,2-DIPALMITOYL-SN-GLYCERO-3-PHOSPHATE / type: ligand / ID: 7 / Number of copies: 1 / Formula: PX6
Molecular weightTheoretical: 647.883 Da
Chemical component information

ChemComp-PX6:
1,2-DIPALMITOYL-SN-GLYCERO-3-PHOSPHATE

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Macromolecule #8: DECANE

MacromoleculeName: DECANE / type: ligand / ID: 8 / Number of copies: 4 / Formula: D10
Molecular weightTheoretical: 142.282 Da
Chemical component information

ChemComp-D10:
DECANE

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Macromolecule #9: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 9 / Number of copies: 6 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Macromolecule #10: GAMMA-AMINO-BUTANOIC ACID

MacromoleculeName: GAMMA-AMINO-BUTANOIC ACID / type: ligand / ID: 10 / Number of copies: 1 / Formula: ABU
Molecular weightTheoretical: 103.12 Da
Chemical component information

ChemComp-ABU:
GAMMA-AMINO-BUTANOIC ACID

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Macromolecule #11: HEXADECANE

MacromoleculeName: HEXADECANE / type: ligand / ID: 11 / Number of copies: 2 / Formula: R16
Molecular weightTheoretical: 226.441 Da
Chemical component information

ChemComp-R16:
HEXADECANE

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Macromolecule #12: HISTAMINE

MacromoleculeName: HISTAMINE / type: ligand / ID: 12 / Number of copies: 2 / Formula: HSM
Molecular weightTheoretical: 111.145 Da
Chemical component information

ChemComp-HSM:
HISTAMINE / neurotransmitter, hormone*YM

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Macromolecule #13: water

MacromoleculeName: water / type: ligand / ID: 13 / Number of copies: 3 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

BufferpH: 7.6
Component:
ConcentrationFormulaName
300.0 mMNaClSodium chloride
50.0 mMHEPES
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 287 K / Instrument: LEICA PLUNGER

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.1 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 130000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: AB INITIO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 37979
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationSoftware - Name: RELION (ver. 3.1)

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

PDB-7qn7:
Cryo-EM structure of human full-length extrasynaptic alpha4beta3delta GABA(A)R in complex with GABA, histamine and nanobody Nb25

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