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- PDB-6i53: Cryo-EM structure of the human synaptic alpha1-beta3-gamma2 GABAA... -

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Basic information

Entry
Database: PDB / ID: 6i53
TitleCryo-EM structure of the human synaptic alpha1-beta3-gamma2 GABAA receptor in complex with Megabody38 in a lipid nanodisc
Components
  • (Gamma-aminobutyric acid receptor subunit ...) x 3
  • Megabody38
KeywordsTRANSPORT PROTEIN / Ligand gated ion channel Cys-Loop receptor GABA-A receptor
Function / homologyNeurotransmitter-gated ion-channel ligand-binding domain / Gamma-aminobutyric-acid A receptor, beta subunit / 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 / Gamma-aminobutyric-acid A receptor, alpha subunit / Neurotransmitter-gated ion-channel, conserved site / Neurotransmitter-gated ion-channel transmembrane domain superfamily ...Neurotransmitter-gated ion-channel ligand-binding domain / Gamma-aminobutyric-acid A receptor, beta subunit / 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 / Gamma-aminobutyric-acid A receptor, alpha subunit / Neurotransmitter-gated ion-channel, conserved site / Neurotransmitter-gated ion-channel transmembrane domain superfamily / 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 receptor activation / Gamma-aminobutyric-acid A receptor, alpha 1 subunit / benzodiazepine receptor activity / inner ear receptor cell development / inhibitory extracellular ligand-gated ion channel activity / GABA-A receptor complex / GABA receptor complex / GABA-A receptor activity / GABA-gated chloride ion channel activity / regulation of postsynaptic membrane potential / cellular response to histamine / synaptic transmission, GABAergic / gamma-aminobutyric acid signaling pathway / innervation / integral component of postsynaptic specialization membrane / 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 / drug binding / 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 sourceHomo sapiens (human)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.2 Å resolution
AuthorsLaverty, D. / Desai, R. / Uchanski, T. / Masiulis, S. / Wojciech, J.S. / Malinauskas, T. / Zivanov, J. / Pardon, E. / Steyaert, J. / Miller, K.W. / Aricescu, A.R.
CitationJournal: Nature / Year: 2019
Title: Cryo-EM structure of the human α1β3γ2 GABA receptor in a lipid bilayer.
Authors: Duncan Laverty / Rooma Desai / Tomasz Uchański / Simonas Masiulis / Wojciech J Stec / Tomas Malinauskas / Jasenko Zivanov / Els Pardon / Jan Steyaert / Keith W Miller / A Radu Aricescu
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Nov 12, 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.name

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Structure visualization

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Assembly

Deposited unit
E: Gamma-aminobutyric acid receptor subunit beta-3
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
G: Megabody38
hetero molecules


Theoretical massNumber of molelcules
Total (without water)294,68040
Polyers285,1076
Non-polymers9,57334
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)40550
ΔGint (kcal/M)-84
Surface area (Å2)72780
MethodPISA

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Components

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

#1: 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): HEK293 / Production host: Homo sapiens (human) / References: UniProt: P28472
#2: Protein/peptide Gamma-aminobutyric acid receptor subunit alpha-1 / GABA(A) receptor subunit alpha-1


Mass: 52916.602 Da / Num. of mol.: 2 / Source: (gene. exp.) Homo sapiens (human) / Gene: GABRA1 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: P14867
#3: Protein/peptide Gamma-aminobutyric acid receptor subunit gamma-2 / GABA(A) receptor subunit gamma-2


Mass: 56922.055 Da / Num. of mol.: 1 / Source: (gene. exp.) Homo sapiens (human) / Gene: GABRG2 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: P18507

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Protein/peptide , 1 types, 1 molecules G

#4: Protein/peptide Megabody38


Mass: 13462.909 Da / Num. of mol.: 1 / Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)

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Non-polymers , 5 types, 34 molecules

#5: Chemical
ChemComp-NAG / N-ACETYL-D-GLUCOSAMINE


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


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


Mass: 180.156 Da / Num. of mol.: 11 / Formula: C6H12O6
#8: Chemical ChemComp-POV / (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate / POPC


Mass: 760.076 Da / Num. of mol.: 3 / Formula: C42H82NO8P / POPC
#9: 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

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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 alpha1-beta3-gamma2 GABA-A receptor in complex with Megabody38 and in lipid nanodiscsCOMPLEX1,2,3,40MULTIPLE SOURCES
2Human alpha1-beta3-gamma2 GABA-A receptorCOMPLEX1,2,31RECOMBINANT
3Megabody38COMPLEX41RECOMBINANT
Molecular weightValue: 0.4 MDa / Experimental value: NO
Source (natural)
IDEntity assembly IDNcbi tax IDOrganism
229606Homo sapiens (human)
339844Lama glama (llama)
Source (recombinant)
IDEntity assembly IDNcbi tax IDOrganismStrain
229606Homo sapiens (human)Embryonic kidney cells
33562Escherichia coli (E. coli)
Buffer solutionpH: 7.2
Buffer component
IDConc.NameFormulaBuffer ID
1100 mMSodium chlorideNaCl1
210 mMHEPESC8H18N2O4S1
SpecimenConc.: 0.5 mg/ml
Details: The purified receptor-nanodisc complex was mixed with Mb38 prior to grid preparation.
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: UltrAuFoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 287.15 kelvins

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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 / Calibrated magnification: 75000 / Nominal defocus max: 700 nm / Nominal defocus min: 500 nm / Calibrated defocus min: 500 nm / Calibrated defocus max: 700 nm / Cs: 2.7 mm / C2 aperture diameter: 50 microns
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 60 sec. / Electron dose: 30.84 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Number of grids imaged: 1 / Number of real images: 784
EM imaging opticsPhase plate: Volta phase plate

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Processing

SoftwareName: PHENIX / Version: 1.14rc2_3191: / Classification: refinement
EM software
IDNameVersionCategory
5Gctfv1.18CTF correction
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
12RELIONv3.0classification
13RELION3.03D reconstruction
14PHENIXmodel refinement
CTF correctionDetails: CTF parameters were estimated using GCTF and CTF correction performed in RELION (full phase and amplitude correction).
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 55449 / Algorithm: FOURIER SPACE / Number of class averages: 1 / Symmetry type: POINT
Atomic model buildingRef protocol: AB INITIO MODEL / Ref space: REAL
RefineStereochemistry target values: CDL v1.2
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004915782
ELECTRON MICROSCOPYf_angle_d0.754321490
ELECTRON MICROSCOPYf_chiral_restr0.04952487
ELECTRON MICROSCOPYf_plane_restr0.00652605
ELECTRON MICROSCOPYf_dihedral_angle_d7.53989236

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