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- PDB-5bkf: Cyro-EM structure of human Glycine Receptor alpha2-beta heteromer... -

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Basic information

Entry
Database: PDB / ID: 5bkf
TitleCyro-EM structure of human Glycine Receptor alpha2-beta heteromer, Glycine bound, desensitized state
Components
  • Glycine receptor subunit alpha-2
  • Glycine receptor subunit beta,Green fluorescent protein
KeywordsMEMBRANE PROTEIN / SIGNALING PROTEIN / glycine receptor / alpha2-beta hetero-pentamer / glycine
Function / homology
Function and homology information


glycine-gated chloride ion channel activity / glycine-gated chloride channel complex / Neurotransmitter receptors and postsynaptic signal transmission / postsynaptic specialization / acrosome reaction / gamma-aminobutyric acid receptor clustering / extracellularly glycine-gated ion channel activity / righting reflex / extracellularly glycine-gated chloride channel activity / synaptic transmission, glycinergic ...glycine-gated chloride ion channel activity / glycine-gated chloride channel complex / Neurotransmitter receptors and postsynaptic signal transmission / postsynaptic specialization / acrosome reaction / gamma-aminobutyric acid receptor clustering / extracellularly glycine-gated ion channel activity / righting reflex / extracellularly glycine-gated chloride channel activity / synaptic transmission, glycinergic / glycinergic synapse / cellular response to ethanol / adult walking behavior / cellular response to zinc ion / glycine binding / startle response / chloride channel complex / neuropeptide signaling pathway / GABA-ergic synapse / response to amino acid / monoatomic ion transport / monoatomic ion transmembrane transport / chloride transmembrane transport / visual perception / bioluminescence / generation of precursor metabolites and energy / transmitter-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / cellular response to amino acid stimulus / transmembrane signaling receptor activity / nervous system development / chemical synaptic transmission / postsynaptic membrane / neuron projection / intracellular membrane-bounded organelle / synapse / dendrite / protein-containing complex binding / metal ion binding / plasma membrane / cytoplasm
Similarity search - Function
Glycine receptor alpha2 / : / : / Glycine receptor beta / Glycine receptor alpha / 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 ...Glycine receptor alpha2 / : / : / Glycine receptor beta / Glycine receptor alpha / 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 / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein
Similarity search - Domain/homology
GLYCINE / Glycine receptor subunit alpha-2 / Green fluorescent protein / Glycine receptor subunit beta
Similarity search - Component
Biological speciesHomo sapiens (human)
Aequorea victoria (jellyfish)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsYu, H. / Wang, W.
Funding support United States, 1items
OrganizationGrant numberCountry
Welch FoundationI-2020-20190330 United States
CitationJournal: Neuron / Year: 2021
Title: Characterization of the subunit composition and structure of adult human glycine receptors.
Authors: Hailong Yu / Xiao-Chen Bai / Weiwei Wang /
Abstract: The strychnine-sensitive pentameric glycine receptor (GlyR) mediates fast inhibitory neurotransmission in the mammalian nervous system. Only heteromeric GlyRs mediate synaptic transmission, as they ...The strychnine-sensitive pentameric glycine receptor (GlyR) mediates fast inhibitory neurotransmission in the mammalian nervous system. Only heteromeric GlyRs mediate synaptic transmission, as they contain the β subunit that permits clustering at the synapse through its interaction with scaffolding proteins. Here, we show that α2 and β subunits assemble with an unexpected 4:1 stoichiometry to produce GlyR with native electrophysiological properties. We determined structures in multiple functional states at 3.6-3.8 Å resolutions and show how 4:1 stoichiometry is consistent with the structural features of α2β GlyR. Furthermore, we show that one single β subunit in each GlyR gives rise to the characteristic electrophysiological properties of heteromeric GlyR, while more β subunits render GlyR non-conductive. A single β subunit ensures a univalent GlyR-scaffold linkage, which means the scaffold alone regulates the cluster properties.
History
DepositionMar 19, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 8, 2021Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: Glycine receptor subunit alpha-2
B: Glycine receptor subunit alpha-2
C: Glycine receptor subunit alpha-2
D: Glycine receptor subunit alpha-2
E: Glycine receptor subunit beta,Green fluorescent protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)247,83519
Polymers245,4695
Non-polymers2,36614
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area27860 Å2
ΔGint-128 kcal/mol
Surface area63470 Å2

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Components

#1: Protein
Glycine receptor subunit alpha-2 /


Mass: 41676.004 Da / Num. of mol.: 4 / Mutation: second cytoplasmic domain deleted
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GLRA2 / Production host: Homo sapiens (human) / References: UniProt: P23416
#2: Protein Glycine receptor subunit beta,Green fluorescent protein /


Mass: 78765.086 Da / Num. of mol.: 1 / Mutation: four substitutions in the GFP portion
Source method: isolated from a genetically manipulated source
Details: This is a GFP insertion between helices M3-M4 of Glycine Receptor Beta
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Aequorea victoria (jellyfish)
Gene: GLRB, GFP / Production host: Homo sapiens (human) / References: UniProt: P48167, UniProt: P42212
#3: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 9
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#4: Chemical
ChemComp-GLY / GLYCINE / Glycine


Type: peptide linking / Mass: 75.067 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C2H5NO2 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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

Component
IDNameTypeEntity IDParent-IDSource
1Glycine receptor alpha2-beta heteromer, glycine bound, desensitized stateCOMPLEX#1-#20MULTIPLE SOURCES
2Glycine receptor alpha2COMPLEX#11RECOMBINANT
3Glycine receptor beta, GFP chimeraCOMPLEX#21RECOMBINANT
Molecular weightValue: 0.257 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Homo sapiens (human)9606
33Aequorea victoria (jellyfish)6100
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Homo sapiens (human)9606
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris1
2200 mMSodium chlorideNaClSodium chloride1
32 mMGlycine1
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 800 nm / Calibrated defocus max: 3200 nm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 80 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5999
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameCategory
1RELIONparticle selection
2SerialEMimage acquisition
4GctfCTF correction
7Cootmodel fitting
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
11RELIONclassification
12RELION3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 555287
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 56419 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model buildingPDB-ID: 7KUY

7kuy

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