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- EMDB-51963: Cryo-EM structure of the human GABAA receptor alpha1 subunit in c... -

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Entry
Database: EMDB / ID: EMD-51963
TitleCryo-EM structure of the human GABAA receptor alpha1 subunit in complex with the assembly factor NACHO/TMEM35A
Map dataPostprocessed, sharpened cryoEM map. For best results (especially for visualising lipids), please convert to MTZ format.
Sample
  • Complex: Complex of Gamma-aminobutyric acid receptor subunit alpha-1 with assembly factor NACHO/TMEM35A
    • Protein or peptide: Gamma-aminobutyric acid receptor subunit alpha-1
    • Protein or peptide: Novel acetylcholine receptor chaperone
  • Ligand: 1,2-DIPALMITOYL-SN-GLYCERO-3-PHOSPHATE
  • Ligand: (7S)-4-HYDROXY-N,N,N-TRIMETHYL-9-OXO-7-[(PALMITOYLOXY)METHYL]-3,5,8-TRIOXA-4-PHOSPHAHEXACOSAN-1-AMINIUM 4-OXIDE
  • Ligand: (1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE
  • Ligand: CHOLESTEROL
KeywordsAssembly intermediate / ion channel / chaperone / Neurotransmitter receptor / Membrane protein biogenesis / MEMBRANE PROTEIN
Function / homology
Function and homology information


acetylcholine receptor regulator activity / GABA receptor complex / GABA receptor activation / positive regulation of protein localization to cell surface / GABA-A receptor activity / GABA-gated chloride ion channel activity / GABA-A receptor complex / inhibitory synapse assembly / peroxisomal membrane / gamma-aminobutyric acid signaling pathway ...acetylcholine receptor regulator activity / GABA receptor complex / GABA receptor activation / positive regulation of protein localization to cell surface / GABA-A receptor activity / GABA-gated chloride ion channel activity / GABA-A receptor complex / inhibitory synapse assembly / peroxisomal membrane / gamma-aminobutyric acid signaling pathway / postsynaptic specialization membrane / synaptic transmission, GABAergic / Signaling by ERBB4 / chloride channel complex / chaperone-mediated protein complex assembly / dendrite membrane / cytoplasmic vesicle membrane / chloride transmembrane transport / transmitter-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / GABA-ergic synapse / cytoplasmic vesicle / dendritic spine / postsynapse / endoplasmic reticulum membrane / endoplasmic reticulum / plasma membrane
Similarity search - Function
Transmembrane protein 35A/B / Gamma-aminobutyric-acid A receptor, alpha 1 subunit / : / Gamma-aminobutyric-acid A receptor, alpha subunit / : / Gamma-aminobutyric acid A receptor/Glycine receptor alpha / Neurotransmitter-gated ion-channel transmembrane domain / Neurotransmitter-gated ion-channel transmembrane region / Neurotransmitter-gated ion-channel, conserved site / Neurotransmitter-gated ion-channels signature. ...Transmembrane protein 35A/B / Gamma-aminobutyric-acid A receptor, alpha 1 subunit / : / Gamma-aminobutyric-acid A receptor, alpha subunit / : / Gamma-aminobutyric acid A receptor/Glycine receptor alpha / Neurotransmitter-gated ion-channel transmembrane domain / Neurotransmitter-gated ion-channel transmembrane region / Neurotransmitter-gated ion-channel, conserved site / Neurotransmitter-gated ion-channels signature. / 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 alpha-1 / Novel acetylcholine receptor chaperone
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsHooda Y / Sente A / Judy RM / Smalinskaite L / Peak-Chew S / Naydenova K / Malinauskas T / Hardwick SW / Chirgadze DY / Aricescu AR / Hegde RS
Funding support United Kingdom, United States, 5 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MC_UP_ A022_1007 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)1R01GM135550-01 United States
Medical Research Council (MRC, United Kingdom)MC_EX_MR/T046279/1 United Kingdom
National Science Foundation (NSF, United States)2014862 United States
CitationJournal: bioRxiv / Year: 2024
Title: Mechanism of NACHO-mediated assembly of pentameric ligand-gated ion channels.
Authors: Yogesh Hooda / Andrija Sente / Ryan M Judy / Luka Smalinskaitė / Sew-Yeu Peak-Chew / Katerina Naydenova / Tomas Malinauskas / Steven W Hardwick / Dimitri Y Chirgadze / A Radu Aricescu / Ramanujan S Hegde /
Abstract: Pentameric ligand-gated ion channels (pLGICs) are cell surface receptors of crucial importance for animal physiology. This diverse protein family mediates the ionotropic signals triggered by major ...Pentameric ligand-gated ion channels (pLGICs) are cell surface receptors of crucial importance for animal physiology. This diverse protein family mediates the ionotropic signals triggered by major neurotransmitters and includes γ-aminobutyric acid receptors (GABARs) and acetylcholine receptors (nAChRs). Receptor function is fine-tuned by a myriad of endogenous and pharmacological modulators. A functional pLGIC is built from five homologous, sometimes identical, subunits, each containing a β-scaffold extracellular domain (ECD), a four-helix transmembrane domain (TMD) and intracellular loops of variable length. Although considerable progress has been made in understanding pLGICs in structural and functional terms, the molecular mechanisms that enable their assembly at the endoplasmic reticulum (ER) in a vast range of potential subunit configurations remain unknown. Here, we identified candidate pLGICs assembly factors selectively associated with an unassembled GABAR subunit. Focusing on one of the candidates, we determined the cryo-EM structure of an assembly intermediate containing two α1 subunits of GABAR each bound to an ER-resident membrane protein NACHO. The structure showed how NACHO shields the principal (+) transmembrane interface of α1 subunits containing an immature extracellular conformation. Crosslinking and structure-prediction revealed an adjacent surface on NACHO for β2 subunit interactions to guide stepwise oligimerisation. Mutations of either subunit-interacting surface on NACHO also impaired the formation of homopentameric α7 nAChRs, pointing to a generic framework for pLGIC assembly. Our work provides the foundation for understanding the regulatory principles underlying pLGIC structural diversity.
History
DepositionOct 30, 2024-
Header (metadata) releaseDec 4, 2024-
Map releaseDec 4, 2024-
UpdateDec 4, 2024-
Current statusDec 4, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_51963.png

Downloads & links

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Map

FileDownload / File: emd_51963.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPostprocessed, sharpened cryoEM map. For best results (especially for visualising lipids), please convert to MTZ format.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.47 Å/pix.
x 256 pix.
= 375.232 Å		1.47 Å/pix.
x 256 pix.
= 375.232 Å		1.47 Å/pix.
x 256 pix.
= 375.232 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.46575 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 0.811
Minimum - Maximum-8.331097 - 11.087415999999999
Average (Standard dev.)0.001480731 (±0.11154089)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 375.232 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_51963_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened cryoEM map from 3D refinement. For best...

Fileemd_51963_additional_1.map
AnnotationUnsharpened cryoEM map from 3D refinement. For best results (especially for visualising lipids), please convert to MTZ format.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: CryoEM half map 2

Fileemd_51963_half_map_1.map
AnnotationCryoEM half map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: CryoEM half map 1

Fileemd_51963_half_map_2.map
AnnotationCryoEM half map 1
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 : Complex of Gamma-aminobutyric acid receptor subunit alpha-1 with ...

EntireName: Complex of Gamma-aminobutyric acid receptor subunit alpha-1 with assembly factor NACHO/TMEM35A
Components
  • Complex: Complex of Gamma-aminobutyric acid receptor subunit alpha-1 with assembly factor NACHO/TMEM35A
    • Protein or peptide: Gamma-aminobutyric acid receptor subunit alpha-1
    • Protein or peptide: Novel acetylcholine receptor chaperone
  • Ligand: 1,2-DIPALMITOYL-SN-GLYCERO-3-PHOSPHATE
  • Ligand: (7S)-4-HYDROXY-N,N,N-TRIMETHYL-9-OXO-7-[(PALMITOYLOXY)METHYL]-3,5,8-TRIOXA-4-PHOSPHAHEXACOSAN-1-AMINIUM 4-OXIDE
  • Ligand: (1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE
  • Ligand: CHOLESTEROL

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Supramolecule #1: Complex of Gamma-aminobutyric acid receptor subunit alpha-1 with ...

SupramoleculeName: Complex of Gamma-aminobutyric acid receptor subunit alpha-1 with assembly factor NACHO/TMEM35A
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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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: Homo sapiens (human)
Molecular weightTheoretical: 54.100066 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGILPSPGMP ALLSLVSLLS VLLMGCVAET GWSHPQFEKG GGSGGGSGGS AWSHPQFEKG GSTGDNTTVF TRILDRLLDG YDNRLRPGL GERVTEVKTD IFVTSFGPVS DHDMEYTIDV FFRQSWKDER LKFKGPMTVL RLNNLMASKI WTPDTFFHNG K KSVAHNMT ...String:
MGILPSPGMP ALLSLVSLLS VLLMGCVAET GWSHPQFEKG GGSGGGSGGS AWSHPQFEKG GSTGDNTTVF TRILDRLLDG YDNRLRPGL GERVTEVKTD IFVTSFGPVS DHDMEYTIDV FFRQSWKDER LKFKGPMTVL RLNNLMASKI WTPDTFFHNG K KSVAHNMT MPNKLLRITE DGTLLYTMRL TVRAECPMHL EDFPMDAHAC PLKFGSYAYT RAEVVYEWTR EPARSVVVAE DG SRLNQYD LLGQTVDSGI VQSSTGEYVV MTTHFHLKRK IGYFVIQTYL PCIMTVILSQ VSFWLNRESV PARTVFGVTT VLT MTTLSI SARNSLPKVA YATAMDWFIA VCYAFVFSAL IEFATVNYFT KRGYAWDGKS VVPEKPKKVK DPLIKKNNTY APTA TSYTP NLARGDPGLA TIAKSATIEP KEVKPETKPP EPKKTFNSVS KIDRLSRIAF PLLFGIFNLV YWATYLNREP QLKAP TPHQ

UniProtKB: Gamma-aminobutyric acid receptor subunit alpha-1

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Macromolecule #2: Novel acetylcholine receptor chaperone

MacromoleculeName: Novel acetylcholine receptor chaperone / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 20.141947 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
MASPRTVTIV ALSVALGLFF VFMGTIKLTP RLSKDAYSEM KRAYKSYVRA LPLLKKMGIN SILLRKSIGA LEVACGIVMT LVPGRPKDV ANFFLLLLVL AVLFFHQLVG DPLKRYAHAL VFGILLTCRL LIARKPEDRS SEKKPLPGNA EEQPSLYEKA P QGKVKVSG GSGGSGGSGK TETSQVAPA

UniProtKB: Novel acetylcholine receptor chaperone

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Macromolecule #4: 1,2-DIPALMITOYL-SN-GLYCERO-3-PHOSPHATE

MacromoleculeName: 1,2-DIPALMITOYL-SN-GLYCERO-3-PHOSPHATE / type: ligand / ID: 4 / Number of copies: 2 / Formula: PX6
Molecular weightTheoretical: 647.883 Da
Chemical component information

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

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Macromolecule #5: (7S)-4-HYDROXY-N,N,N-TRIMETHYL-9-OXO-7-[(PALMITOYLOXY)METHYL]-3,5...

MacromoleculeName: (7S)-4-HYDROXY-N,N,N-TRIMETHYL-9-OXO-7-[(PALMITOYLOXY)METHYL]-3,5,8-TRIOXA-4-PHOSPHAHEXACOSAN-1-AMINIUM 4-OXIDE
type: ligand / ID: 5 / Number of copies: 2 / Formula: PC7
Molecular weightTheoretical: 763.1 Da
Chemical component information

ChemComp-PC7:
(7S)-4-HYDROXY-N,N,N-TRIMETHYL-9-OXO-7-[(PALMITOYLOXY)METHYL]-3,5,8-TRIOXA-4-PHOSPHAHEXACOSAN-1-AMINIUM 4-OXIDE / phospholipid*YM

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Macromolecule #6: (1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-...

MacromoleculeName: (1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE
type: ligand / ID: 6 / Number of copies: 2 / Formula: PGT
Molecular weightTheoretical: 751.023 Da
Chemical component information

ChemComp-PGT:
(1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE / phospholipid*YM

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Macromolecule #7: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 7 / Number of copies: 2 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL

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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:
ConcentrationNameFormula
50.0 mMHEPES
300.0 mMsodium chlorideNaCl
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Details: PELCO easiGlow, 30 mA for 30 s
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 287.15 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 23057 / Average electron dose: 45.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.6 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 10530693
Startup modelType of model: OTHER / Details: Ab initio model
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2) / Number images used: 304241
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 3.3.2)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

PDB-9h9e:
Cryo-EM structure of the human GABAA receptor alpha1 subunit in complex with the assembly factor NACHO/TMEM35A

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