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- PDB-9h9e: Cryo-EM structure of the human GABAA receptor alpha1 subunit in c... -

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Basic information

Entry
Database: PDB / ID: 9h9e
TitleCryo-EM structure of the human GABAA receptor alpha1 subunit in complex with the assembly factor NACHO/TMEM35A
Components
  • Gamma-aminobutyric acid receptor subunit alpha-1
  • Novel acetylcholine receptor chaperone
KeywordsMEMBRANE PROTEIN / Assembly intermediate / ion channel / chaperone / Neurotransmitter receptor / Membrane protein biogenesis
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
CHOLESTEROL / Chem-PC7 / Chem-PGT / 1,2-DIPALMITOYL-SN-GLYCERO-3-PHOSPHATE / Gamma-aminobutyric acid receptor subunit alpha-1 / Novel acetylcholine receptor chaperone
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsHooda, Y. / Sente, A. / Judy, R.M. / Smalinskaite, L. / Peak-Chew, S. / Naydenova, K. / Malinauskas, T. / Hardwick, S.W. / Chirgadze, D.Y. / Aricescu, A.R. / Hegde, R.S.
Funding support United Kingdom, United States, 5items
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, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 4, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Gamma-aminobutyric acid receptor subunit alpha-1
B: Novel acetylcholine receptor chaperone
C: Novel acetylcholine receptor chaperone
D: Gamma-aminobutyric acid receptor subunit alpha-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)155,40314
Polymers148,4844
Non-polymers6,91910
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "A" or chain "a"
d_2ens_1chain "D" or chain "d"
d_1ens_2chain "C"
d_2ens_2chain "B"
d_1ens_3chain "E"
d_2ens_3chain "F"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
d_11ens_1ASPASPARGARGAA10 - 4181 - 334
d_12ens_1NAGNAGNAGNAGaE1
d_13ens_1NAGNAGNAGNAGaE2
d_14ens_1BMABMABMABMAaE3
d_15ens_1MANMANMANMANaE4
d_16ens_1MANMANMANMANaE5
d_21ens_1ASPASPARGARGDD10 - 4181 - 334
d_22ens_1NAGNAGNAGNAGdF1
d_23ens_1NAGNAGNAGNAGdF2
d_24ens_1BMABMABMABMAdF3
d_25ens_1MANMANMANMANdF4
d_26ens_1MANMANMANMANdF5
d_11ens_2ALAALALYSLYSCC2 - 1341 - 133
d_21ens_2ALAALALYSLYSBB2 - 1341 - 133
d_11ens_3PX6PX6PX6PX6A - EG501
d_12ens_3PC7PPCPC7PPCEH502
d_13ens_3PGTLIGPGTLIGEI503
d_14ens_3CLRCLRCLRCLREJ504
d_21ens_3PX6PX6PX6PX6FK501
d_22ens_3PC7PPCPC7PPCFL502
d_23ens_3PGTLIGPGTLIGFM503
d_24ens_3CLRCLRCLRCLRFN504

NCS ensembles :
ID
ens_1
ens_2
ens_3

NCS oper:
IDCodeMatrixVector
1given(-0.999905676033, 0.0137333665908, 0.000183517285843), (-0.0137339250019, -0.999899558393, -0.00350034715486), (0.000135427302399, -0.00350253740087, 0.999993856927)372.757892682, 378.550958102, 0.603190115384
2given(-0.999997340619, 0.00112112961519, -0.00201539670774), (-0.00111973262709, -0.999999132175, -0.000694152643986), (-0.00201617319382, -0.000691894092519, 0.999997728162)375.386150621, 375.591794261, 0.402109569621
3given(-0.999989974895, 0.00441874016191, 0.000724461513131), (-0.00441848729547, -0.999990177092, 0.00035027031286), (0.000726002150311, 0.000347065777372, 0.999999676233)374.376919459, 376.042134534, -0.175160335914

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Components

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Protein , 2 types, 4 molecules ADBC

#1: Protein Gamma-aminobutyric acid receptor subunit alpha-1 / GABA(A) receptor subunit alpha-1


Mass: 54100.066 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GABRA1 / Cell line (production host): Expi293 / Production host: Homo sapiens (human) / References: UniProt: P14867
#2: Protein Novel acetylcholine receptor chaperone


Mass: 20141.947 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TMEM35A / Cell line (production host): Expi293 / Production host: Homo sapiens (human) / References: UniProt: Q53FP2

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Sugars , 1 types, 2 molecules

#3: Polysaccharide alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-3)-2-acetamido-2- ...alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-3)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 910.823 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3[DManpa1-6]DManpb1-3DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/3,5,4/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3/a4-b1_b3-c1_c3-d1_c6-e1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(3+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}[(6+1)][a-D-Manp]{}}}}}LINUCSPDB-CARE

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Non-polymers , 4 types, 8 molecules

#4: Chemical ChemComp-PX6 / 1,2-DIPALMITOYL-SN-GLYCERO-3-PHOSPHATE


Mass: 647.883 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C35H68O8P
#5: Chemical ChemComp-PC7 / (7S)-4-HYDROXY-N,N,N-TRIMETHYL-9-OXO-7-[(PALMITOYLOXY)METHYL]-3,5,8-TRIOXA-4-PHOSPHAHEXACOSAN-1-AMINIUM 4-OXIDE / PHOSPHATIDYLCHOLINE / 1-PALMITOYL-2-STEAROYL-PC


Mass: 763.100 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C42H85NO8P / Comment: phospholipid*YM
#6: Chemical ChemComp-PGT / (1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE / PHOSPHATIDYLGLYCEROL / 1-PALMITOYL-2-OLEOYL-SN-GLYCERO-3-[PHOSPHO-RAC-(1-GLYCEROL)](SODIUM SALT)


Mass: 751.023 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C40H79O10P / Comment: phospholipid*YM
#7: Chemical ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C27H46O

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Details

Has ligand of interestN
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

ComponentName: Complex of Gamma-aminobutyric acid receptor subunit alpha-1 with assembly factor NACHO/TMEM35A
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: Expi293
Buffer solutionpH: 7.6
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPES1
2300 mMsodium chlorideNaCl1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: PELCO easiGlow, 30 mA for 30 s / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 287.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 2600 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 45 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 23057
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1Warp1.07particle selection
2EPUimage acquisition
4CTFFIND4.1.1354CTF correction
7Coot0.9.8.5model fitting
9cryoSPARC3.3.2initial Euler assignment
10cryoSPARC3.3.2final Euler assignment
11cryoSPARC3.3.2classification
12cryoSPARC3.3.23D reconstruction
13PHENIXdev-5430-0000model refinement
14PHENIX1.19.2model refinement
15REFMAC5.8.0258model refinement
16Servalcatmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 10530693
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 304241 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 44.47 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00268116
ELECTRON MICROSCOPYf_angle_d0.426110960
ELECTRON MICROSCOPYf_chiral_restr0.03841290
ELECTRON MICROSCOPYf_plane_restr0.00311308
ELECTRON MICROSCOPYf_dihedral_angle_d8.56793142
Refine LS restraints NCS
Ens-IDDom-IDAsym-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AAELECTRON MICROSCOPYNCS constraints7.7085250771E-12
ens_2d_2CCELECTRON MICROSCOPYNCS constraints3.23054928142E-13
ens_3d_2GAELECTRON MICROSCOPYNCS constraints3.16366584913E-13

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