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- PDB-6hug: CryoEM structure of human full-length alpha1beta3gamma2L GABA(A)R... -

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Basic information

Entry
Database: PDB / ID: 6hug
TitleCryoEM structure of human full-length alpha1beta3gamma2L GABA(A)R in complex with picrotoxin and megabody Mb38.
Components
  • (Gamma-aminobutyric acid receptor subunit ...) x 3
  • Megabody Mb38
KeywordsMEMBRANE PROTEIN / GABAAR / PTX / Membrane / Channel / Nanobody / Megabody / Cys-loop / PLGIC / Inhibition / Signalling / CNS / Neurons / Chloride / Ion / GABA / Picrotoxin
Function / homologyNeurotransmitter-gated ion-channel, conserved site / Neurotransmitter-gated ion-channel transmembrane domain superfamily / Gamma-aminobutyric-acid A receptor, gamma subunit / Gamma-aminobutyric-acid A receptor, gamma 2 subunit / Gamma-aminobutyric acid A receptor/Glycine receptor alpha / Neurotransmitter-gated ion-channel transmembrane domain / Neurotransmitter-gated ion-channel / Neurotransmitter-gated ion-channel ligand-binding domain / Gamma-aminobutyric-acid A receptor, alpha subunit / Gamma-aminobutyric-acid A receptor, alpha 1 subunit ...Neurotransmitter-gated ion-channel, conserved site / Neurotransmitter-gated ion-channel transmembrane domain superfamily / Gamma-aminobutyric-acid A receptor, gamma subunit / Gamma-aminobutyric-acid A receptor, gamma 2 subunit / Gamma-aminobutyric acid A receptor/Glycine receptor alpha / Neurotransmitter-gated ion-channel transmembrane domain / Neurotransmitter-gated ion-channel / Neurotransmitter-gated ion-channel ligand-binding domain / Gamma-aminobutyric-acid A receptor, alpha subunit / Gamma-aminobutyric-acid A receptor, alpha 1 subunit / Gamma-aminobutyric-acid A receptor, beta subunit / Neurotransmitter-gated ion-channel ligand-binding domain superfamily / Neurotransmitter-gated ion-channel ligand binding domain / Neurotransmitter-gated ion-channel transmembrane region / Neurotransmitter-gated ion-channels signature. / GABA A receptor activation / GABA A receptor activation / GABA receptor activation / benzodiazepine receptor activity / inner ear receptor cell development / inhibitory extracellular ligand-gated ion channel activity / GABA-A receptor complex / GABA-A receptor activity / GABA-gated chloride ion channel activity / regulation of postsynaptic membrane potential / synaptic transmission, GABAergic / cellular response to histamine / gamma-aminobutyric acid signaling pathway / innervation / inhibitory postsynaptic potential / chloride channel activity / adult behavior / chloride channel complex / chloride transmembrane transport / cochlea development / dendrite membrane / GABA-ergic synapse / nervous system process / roof of mouth development / transmitter-gated ion channel activity involved in regulation of postsynaptic membrane potential / ion transmembrane transport / regulation of membrane potential / post-embryonic development / sensory perception of sound / cytoplasmic vesicle membrane / postsynapse / postsynaptic membrane / chemical synaptic transmission / negative regulation of neuron apoptotic process / cell junction / neuron projection / synapse / axon / integral component of plasma membrane / signal transduction / identical protein binding / plasma membrane / Gamma-aminobutyric acid receptor subunit alpha-1 / Gamma-aminobutyric acid receptor subunit gamma-2 / Gamma-aminobutyric acid receptor subunit beta-3
Function and homology information
Specimen sourceBos taurus (cattle)
Homo sapiens (human)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.1 Å resolution
AuthorsMasiulis, S. / Desai, R. / Uchanski, T. / Serna Martin, I. / Laverty, D. / Karia, D. / Malinauskas, T. / Jasenko, Z. / Pardon, E. / Kotecha, A. / Steyaert, J. / Miller, K.W. / Aricescu, A.R.
CitationJournal: Nature / Year: 2019
Title: GABA receptor signalling mechanisms revealed by structural pharmacology.
Authors: Simonas Masiulis / Rooma Desai / Tomasz Uchański / Itziar Serna Martin / Duncan Laverty / Dimple Karia / Tomas Malinauskas / Jasenko Zivanov / Els Pardon / Abhay Kotecha / Jan Steyaert / Keith W Miller / A Radu Aricescu
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Oct 8, 2018 / Release: Jan 2, 2019
RevisionDateData content typeGroupCategoryItemProviderType
1.0Jan 2, 2019Structure modelrepositoryInitial release
1.1Jan 16, 2019Structure modelData collection / Database referencescitation / citation_author_citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

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Assembly

Deposited unit
A: Gamma-aminobutyric acid receptor subunit alpha-1
B: Gamma-aminobutyric acid receptor subunit beta-3
C: Gamma-aminobutyric acid receptor subunit gamma-2
D: Gamma-aminobutyric acid receptor subunit alpha-1
E: Gamma-aminobutyric acid receptor subunit beta-3
G: Megabody Mb38
hetero molecules


Theoretical massNumber of molelcules
Total (without water)331,42541
Polyers323,3006
Non-polymers8,12535
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)41570
ΔGint (kcal/M)-79
Surface area (Å2)72630
MethodPISA

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Components

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Gamma-aminobutyric acid receptor subunit ... , 3 types, 5 molecules ADBEC

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


Mass: 49852.090 Da / Num. of mol.: 2
Details: Potential signal peptide: MKKSPGLSDY LWAWTLFLST LTGRSYG FLAG tag: DYKDDDDK
Source: (gene. exp.) Bos taurus (cattle) / Gene: GABRA1 / Cell line (production host): HEK293SGnTI- / Production host: Homo sapiens (human) / References: UniProt: P08219
#2: Protein/peptide Gamma-aminobutyric acid receptor subunit beta-3 / GABA(A) receptor subunit beta-3


Mass: 54444.578 Da / Num. of mol.: 2 / Source: (gene. exp.) Homo sapiens (human) / Gene: GABRB3 / Cell line (production host): HEK293SGnTI- / Production host: Homo sapiens (human) / References: UniProt: P28472
#3: Protein/peptide Gamma-aminobutyric acid receptor subunit gamma-2 / GABA(A) receptor subunit gamma-2


Mass: 56922.055 Da / Num. of mol.: 1 / Details: Linker sequence: GGSGGSGGSGK 1D4 tag: TETSQVAPA / Source: (gene. exp.) Homo sapiens (human) / Gene: GABRG2 / Cell line (production host): HEK293SGnTI- / Production host: Homo sapiens (human) / References: UniProt: P18507

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Protein/peptide , 1 types, 1 molecules G

#4: Protein/peptide Megabody Mb38


Mass: 57784.301 Da / Num. of mol.: 1 / Details: 6His tag: HHHHHH EPEA tag: EPEA / Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)

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Non-polymers , 5 types, 35 molecules

#5: Chemical 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 / dioctanoyl l-alpha-phosphatidyl-d-myo-inositol 4,5-diphosphate


Mass: 746.566 Da / Num. of mol.: 2 / Formula: C25H49O19P3
#6: Chemical
ChemComp-NAG / N-ACETYL-D-GLUCOSAMINE


Mass: 221.208 Da / Num. of mol.: 14 / Formula: C8H15NO6 / N-Acetylglucosamine
#7: Chemical
ChemComp-BMA / BETA-D-MANNOSE


Mass: 180.156 Da / Num. of mol.: 4 / Formula: C6H12O6
#8: Chemical
ChemComp-MAN / ALPHA-D-MANNOSE


Mass: 180.156 Da / Num. of mol.: 14 / Formula: C6H12O6
#9: Chemical ChemComp-RI5 / (1aR,2aR,3S,6R,6aS,8aS,8bR,9R)-2a-hydroxy-8b-methyl-9-(prop-1-en-2-yl)hexahydro-3,6-methano-1,5,7-trioxacyclopenta[ij]cyclopropa[a]azulene-4,8(3H)-dione / PICROTOXIN


Mass: 292.284 Da / Num. of mol.: 1 / Formula: C15H16O6 / Picrotoxin

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent IDSource
1Human full-length heteromeric alpha1beta3gamma2L GABA(A)R in complex with picrotoxin and megabody Mb38.COMPLEX1,2,3,40MULTIPLE SOURCES
2Human full-length heteromeric alpha1beta3gamma2L GABA(A)R in complex with picrotoxin and megabody Mb38.COMPLEX1,2,31RECOMBINANT
3Human full-length heteromeric alpha1beta3gamma2L GABA(A)R in complex with picrotoxin and megabody Mb38.COMPLEX41RECOMBINANT
Molecular weightValue: 0.33 MDa / Experimental value: NO
Source (natural)
IDEntity assembly IDCellular locationNcbi tax IDOrganOrganism
22Plasma membrane9606BrainHomo sapiens (human)
339844Lama glama (llama)
Source (recombinant)
IDEntity assembly IDCellNcbi tax IDOrganism
22HEK293S9606Homo sapiens (human)
33562Escherichia coli (E. coli)
Buffer solutionpH: 7.6
SpecimenConc.: 0.1 mg/ml / Details: Monodisperse sample / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 3.5 ul of 0.1 mg/ml protein solution was applied on a grid in the Vitrobot MkIV chamber set to 95% RH at 14.5 degC for 30s and then blotted for 5.5 s and plunged
Grid material: GOLD / Grid mesh size: 300 / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: 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: 75000 / Nominal defocus max: 700 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 30 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)
EM imaging opticsPhase plate: Volta phase plate FEI company.

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Processing

EM software
IDNameVersionCategoryDetails
2EPUimage acquisition
4Gctf1.08CTF correction
7UCSF Chimeramodel fitting
9RELION3.0initial Euler assignmentRefine3D
10RELION3.0final Euler assignmentRefine3D
11RELION3.0classificationClass3D
12RELION3.03D reconstructionRefine3D
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 56269 / Symmetry type: POINT
Atomic model buildingRef protocol: FLEXIBLE FIT

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