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- PDB-6dw1: Cryo-EM structure of the benzodiazepine-sensitive alpha1beta1gamm... -

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Basic information

Entry
Database: PDB / ID: 6dw1
TitleCryo-EM structure of the benzodiazepine-sensitive alpha1beta1gamma2S tri-heteromeric GABAA receptor in complex with GABA (ECD map)
Components(Gamma-aminobutyric acid receptor subunit ...) x 3
KeywordsMEMBRANE PROTEIN / Neurotransmission / GABA Receptors / GABA / Cys Loop Receptors / Ion Channel
Function / homologyGamma-aminobutyric acid A receptor/Glycine receptor alpha / Neurotransmitter-gated ion-channel ligand-binding domain superfamily / Gamma-aminobutyric-acid A receptor, gamma 2 subunit / Gamma-aminobutyric-acid A receptor, gamma subunit / Gamma-aminobutyric-acid A receptor, alpha 1 subunit / Gamma-aminobutyric-acid A receptor, beta subunit / Gamma-aminobutyric-acid A receptor, alpha subunit / Neurotransmitter-gated ion-channel / Neurotransmitter-gated ion-channel transmembrane domain / Neurotransmitter-gated ion-channel, conserved site ...Gamma-aminobutyric acid A receptor/Glycine receptor alpha / Neurotransmitter-gated ion-channel ligand-binding domain superfamily / Gamma-aminobutyric-acid A receptor, gamma 2 subunit / Gamma-aminobutyric-acid A receptor, gamma subunit / Gamma-aminobutyric-acid A receptor, alpha 1 subunit / Gamma-aminobutyric-acid A receptor, beta subunit / Gamma-aminobutyric-acid A receptor, alpha subunit / Neurotransmitter-gated ion-channel / Neurotransmitter-gated ion-channel transmembrane domain / Neurotransmitter-gated ion-channel, conserved site / Neurotransmitter-gated ion-channel transmembrane domain superfamily / Neurotransmitter-gated ion-channel ligand-binding domain / Neurotransmitter-gated ion-channel transmembrane region / GABA A receptor activation / Neurotransmitter-gated ion-channels signature. / Neurotransmitter-gated ion-channel ligand binding domain / anion channel activity / inhibitory extracellular ligand-gated ion channel activity / benzodiazepine receptor activity / GABA-gated chloride ion channel activity / GABA-A receptor complex / GABA-A receptor activity / transmitter-gated ion channel activity involved in regulation of postsynaptic membrane potential / GABA receptor complex / cellular response to histamine / synaptic transmission, GABAergic / inhibitory synapse / gamma-aminobutyric acid signaling pathway / GABA receptor binding / chloride transport / chloride channel activity / chloride channel complex / central nervous system neuron development / dendrite membrane / ligand-gated ion channel activity / ovulation cycle / response to progesterone / ion transport / response to toxic substance / cytoplasmic vesicle membrane / nuclear envelope / postsynaptic membrane / receptor complex / drug binding / cell junction / dendrite / integral component of plasma membrane / membrane / plasma membrane / nucleus / cytoplasm / Gamma-aminobutyric acid receptor subunit beta-1 / Gamma-aminobutyric acid receptor subunit gamma-2 / Gamma-aminobutyric acid receptor subunit alpha-1
Function and homology information
Specimen sourceRattus norvegicus (Norway rat)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.1 Å resolution
AuthorsPhulera, S. / Zhu, H. / Yu, J. / Yoshioka, C. / Gouaux, E.
CitationJournal: Elife / Year: 2018
Title: Cryo-EM structure of the benzodiazepine-sensitive α1β1γ2S tri-heteromeric GABA receptor in complex with GABA.
Authors: Swastik Phulera / Hongtao Zhu / Jie Yu / Derek P Claxton / Nate Yoder / Craig Yoshioka / Eric Gouaux
Abstract: Fast inhibitory neurotransmission in the mammalian nervous system is largely mediated by GABA receptors, chloride-selective members of the superfamily of pentameric Cys-loop receptors. Native GABA ...Fast inhibitory neurotransmission in the mammalian nervous system is largely mediated by GABA receptors, chloride-selective members of the superfamily of pentameric Cys-loop receptors. Native GABA receptors are heteromeric assemblies sensitive to many important drugs, from sedatives to anesthetics and anticonvulsant agents, with mutant forms of GABA receptors implicated in multiple neurological diseases. Despite the profound importance of heteromeric GABA receptors in neuroscience and medicine, they have proven recalcitrant to structure determination. Here we present the structure of a tri-heteromeric α1β1γ2S GABA receptor in complex with GABA, determined by single particle cryo-EM at 3.1-3.8 Å resolution, elucidating molecular principles of receptor assembly and agonist binding. Remarkable N-linked glycosylation on the α1 subunit occludes the extracellular vestibule of the ion channel and is poised to modulate receptor assembly and perhaps ion channel gating. Our work provides a pathway to structural studies of heteromeric GABA receptors and a framework for rational design of novel therapeutic agents.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Jun 26, 2018 / Release: Aug 8, 2018

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)242,68029
Polyers238,0955
Non-polymers4,58524
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)20450
ΔGint (kcal/M)8
Surface area (Å2)44730
MethodPISA

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Components

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

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


Mass: 56894.949 Da / Num. of mol.: 1 / Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Gabrg2 / Production host: Homo sapiens (human) / References: UniProt: P18508
#2: Protein/peptide Gamma-aminobutyric acid receptor subunit alpha-1,Gamma-aminobutyric acid receptor subunit alpha-1 / GABA(A) receptor subunit alpha-1


Mass: 46267.105 Da / Num. of mol.: 2 / Fragment: residues 39-334 / Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Gabra1, Gabra-1 / Production host: Homo sapiens (human) / References: UniProt: P62813
#3: Protein/peptide Gamma-aminobutyric acid receptor subunit beta-1,Gamma-aminobutyric acid receptor subunit beta-1 / GABA(A) receptor subunit beta-1


Mass: 44333.012 Da / Num. of mol.: 2 / Fragment: residues 33-474 / Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Gabrb1, Gabrb-1 / Production host: Homo sapiens (human) / References: UniProt: P15431

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Non-polymers , 4 types, 24 molecules

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


Mass: 221.208 Da / Num. of mol.: 12 / Formula: C8H15NO6 / N-Acetylglucosamine
#5: Chemical
ChemComp-BMA / BETA-D-MANNOSE


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


Mass: 180.156 Da / Num. of mol.: 5 / Formula: C6H12O6
#7: Chemical ChemComp-ABU / GAMMA-AMINO-BUTANOIC ACID / GAMMA(AMINO)-BUTYRIC ACID


Mass: 103.120 Da / Num. of mol.: 3 / Formula: C4H9NO2

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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: Triheteromeric alpha1-beta1-gamma2 GABAA receptor / Type: COMPLEX / Details: In complex with alpha1 specific Fab / Entity ID: 1, 2, 3 / Source: RECOMBINANT
Molecular weightUnits: KILODALTONS/NANOMETER / Experimental value: NO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Source (recombinant)Cell: TSA201 / Organism: Homo sapiens (human)
Buffer solutionpH: 7.3
Buffer component
IDConc.NameFormulaBuffer ID
120 mMHEPESC8H18N2O4S1
2200 mMsodium chlorideNaCl1
31 mMDodecyl MaltosideC24H46O111
41.5 mMgamma-Aminobutyric acidC4H9NO21
SpecimenConc.: 0.15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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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: 120000 / Nominal defocus max: -2500 nm / Nominal defocus min: -1200 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 40 sec. / Electron dose: 37 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Number of grids imaged: 1 / Number of real images: 1391

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Processing

EM software
IDNameVersionCategory
1DoG Pickerparticle selection
2EPUimage acquisition
4Gctf1.06CTF correction
5RELION2.1CTF correction
8Coot0.8.6.1model fitting
10PHENIX1.13model refinement
11cryoSPARCinitial Euler assignment
12RELION2.1final Euler assignment
14RELION2.13D reconstruction
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: 68499 / Symmetry type: POINT
Atomic model buildingRef protocol: FLEXIBLE FIT / Ref space: REAL / Target criteria: 0.74

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