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- PDB-6d6u: Human GABA-A receptor alpha1-beta2-gamma2 subtype in complex with... -

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Basic information

Entry
Database: PDB / ID: 6d6u
TitleHuman GABA-A receptor alpha1-beta2-gamma2 subtype in complex with GABA and flumazenil, conformation A
Components
  • (Gamma-aminobutyric acid receptor subunit ...) x 3
  • IgG2b Fab Heavy Chain
  • Kappa Fab Light Chain
KeywordsTRANSPORT PROTEIN / Ligand-gated ion channel / GABA-A receptor / Cys-loop receptor
Function / homologyNeurotransmitter-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, 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, conserved site / Neurotransmitter-gated ion-channel transmembrane domain superfamily ...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, 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, 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. / Neurotransmitter receptors and postsynaptic signal transmission / GABA A receptor activation / Gamma-aminobutyric-acid A receptor, beta subunit / inhibitory extracellular ligand-gated ion channel activity / benzodiazepine receptor activity / inner ear receptor cell development / GABA-gated chloride ion channel activity / GABA-A receptor activity / GABA-A receptor complex / transmitter-gated ion channel activity involved in regulation of postsynaptic membrane potential / GABA receptor complex / cellular response to histamine / synaptic transmission, GABAergic / gamma-aminobutyric acid signaling pathway / innervation / chloride channel activity / adult behavior / chloride channel complex / dendrite membrane / chloride transmembrane transport / cochlea development / extracellular ligand-gated ion channel activity / post-embryonic development / sensory perception of sound / cytoplasmic vesicle membrane / chemical synaptic transmission / postsynaptic membrane / drug binding / negative regulation of neuron apoptotic process / cell junction / axon / integral component of plasma membrane / extracellular exosome / plasma membrane / cytosol / Gamma-aminobutyric acid receptor subunit alpha-1 / Gamma-aminobutyric acid receptor subunit gamma-2 / Gamma-aminobutyric acid receptor subunit beta-2
Function and homology information
Specimen sourceHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.92 Å resolution
AuthorsZhu, S. / Noviello, C.M. / Teng, J. / Walsh Jr, R.M. / Kim, J.J. / Hibbs, R.E.
CitationJournal: Nature / Year: 2018
Title: Structure of a human synaptic GABA receptor.
Authors: Shaotong Zhu / Colleen M Noviello / Jinfeng Teng / Richard M Walsh / Jeong Joo Kim / Ryan E Hibbs
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Apr 22, 2018 / Release: Jun 27, 2018
RevisionDateData content typeGroupCategoryItemProviderType
1.0Jun 27, 2018Structure modelrepositoryInitial release
1.1Jul 11, 2018Structure modelData collection / Database referencescitation / citation_author_citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
1.2Jul 18, 2018Structure modelData collection / Database referencescitation_citation.journal_volume / _citation.page_first / _citation.page_last

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Assembly

Deposited unit
A: Gamma-aminobutyric acid receptor subunit beta-2,Gamma-aminobutyric acid receptor subunit beta-2
B: Gamma-aminobutyric acid receptor subunit alpha-1,Gamma-aminobutyric acid receptor subunit alpha-1
C: Gamma-aminobutyric acid receptor subunit beta-2,Gamma-aminobutyric acid receptor subunit beta-2
D: Gamma-aminobutyric acid receptor subunit alpha-1,Gamma-aminobutyric acid receptor subunit alpha-1
E: Gamma-aminobutyric acid receptor subunit gamma-2,Gamma-aminobutyric acid receptor subunit gamma-2
I: Kappa Fab Light Chain
J: IgG2b Fab Heavy Chain
L: Kappa Fab Light Chain
K: IgG2b Fab Heavy Chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)363,69845
Polyers353,1549
Non-polymers10,54436
Water0
1


  • idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, Gel filtration used to produce homogeneous sample of GABA-A receptor:Fab complex
  • Download structure data
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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

#1: Protein/peptide Gamma-aminobutyric acid receptor subunit beta-2,Gamma-aminobutyric acid receptor subunit beta-2 / GABA(A) receptor subunit beta-2


Mass: 39521.691 Da / Num. of mol.: 2 / Source: (gene. exp.) Homo sapiens (human) / Gene: GABRB2 / Plasmid name: pEZT-BM / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / Variant (production host): GnTI- / References: UniProt: P47870
#2: Protein/peptide Gamma-aminobutyric acid receptor subunit alpha-1,Gamma-aminobutyric acid receptor subunit alpha-1 / GABA(A) receptor subunit alpha-1


Mass: 41061.211 Da / Num. of mol.: 2 / Source: (gene. exp.) Homo sapiens (human) / Gene: GABRA1 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / Variant (production host): GnTI- / References: UniProt: P14867
#3: Protein/peptide Gamma-aminobutyric acid receptor subunit gamma-2,Gamma-aminobutyric acid receptor subunit gamma-2 / GABA(A) receptor subunit gamma-2


Mass: 45354.660 Da / Num. of mol.: 1 / Source: (gene. exp.) Homo sapiens (human) / Gene: GABRG2 / Plasmid name: pEZT-BM / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / Variant (production host): GnTI- / References: UniProt: P18507

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Protein/peptide , 2 types, 4 molecules ILJK

#4: Protein/peptide Kappa Fab Light Chain


Mass: 23505.943 Da / Num. of mol.: 2 / Source: (natural) Mus musculus (house mouse) / Plasmid details: From generated Hybrido / Strain: BALB/c
#5: Protein/peptide IgG2b Fab Heavy Chain


Mass: 49811.043 Da / Num. of mol.: 2 / Source: (natural) Mus musculus (house mouse) / Strain: BALB/c

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Non-polymers , 6 types, 36 molecules

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


Mass: 221.208 Da / Num. of mol.: 10 / Formula: C8H15NO6 / N-Acetylglucosamine
#7: Chemical
ChemComp-BMA / BETA-D-MANNOSE


Mass: 180.156 Da / Num. of mol.: 4 / Formula: C6H12O6
#8: Chemical ChemComp-ABU / GAMMA-AMINO-BUTANOIC ACID / GAMMA(AMINO)-BUTYRIC ACID


Mass: 103.120 Da / Num. of mol.: 2 / Formula: C4H9NO2
#9: Chemical
ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 12 / Formula: C31H50O4
#10: Chemical
ChemComp-MAN / ALPHA-D-MANNOSE


Mass: 180.156 Da / Num. of mol.: 7 / Formula: C6H12O6
#11: Chemical ChemComp-FYP / ethyl 8-fluoro-5-methyl-6-oxo-5,6-dihydro-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate


Mass: 303.288 Da / Num. of mol.: 1 / Formula: C15H14FN3O3

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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

ComponentName: Human GABA-A receptor alpha1-beta2-gamma2 subtype in complex with GABA and flumazenil, conformation A
Type: COMPLEX
Details: Fab fragments generated by proteolytic cleavage of IgG antibody
Entity ID: 1,2,3,4,5 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.348 MDa
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer ID
1150 mMSodium chlorideNaCI1
220 mMTris BaseC4H11NO31
31 mMn-Dodecyl beta-D-maltosideC24H46O111
40.2 mMCholesteryl hemisuccinateC31H50O41
52 mMgamma-Aminobutyric acidNH2(CH2)3COOH1
61 uMFlumazenilC15H14FN3O31
SpecimenConc.: 6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Sample was glow discharged at 30 mA for 80 seconds using a PELCO easiGLow
Grid material: GOLD / Grid mesh size: 200 / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 kelvins / Details: 3 seconds blot time

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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 / Calibrated magnification: 46730 / Nominal defocus max: 4500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 90 kelvins / Temperature (min): 80 kelvins
Image recordingAverage exposure time: 10 sec. / Electron dose: 47 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 2 / Number of real images: 5594
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter upper: 10 eV / Energyfilter lower: -10 eV

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
4Gctf1.06CTF correction
7Coot0.8.9model fitting
9RELION2.1initial Euler assignment
10RELION2.1final Euler assignment
11RELION2.1classification
12RELION2.13D reconstruction
13PHENIX1.13-2988model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNumber of particles selected: 1050737
SymmetryPoint symmetry: C1
3D reconstructionResolution: 3.92 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 292662 / Algorithm: BACK PROJECTION / Number of class averages: 5 / Symmetry type: POINT
Atomic model buildingRef protocol: AB INITIO MODEL / Ref space: REAL
Least-squares processHighest resolution: 3.8 Å

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