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

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Basic information

Entry
Database: PDB / ID: 6hup
TitleCryoEM structure of human full-length alpha1beta3gamma2L GABA(A)R in complex with diazepam (Valium), GABA and megabody Mb38.
Components
  • (Gamma-aminobutyric acid receptor subunit ...) x 3
  • Megabody Mb38
KeywordsMEMBRANE PROTEIN / GABAAR / Membrane protein / Cys-loop / PLGIC
Function / homologyNeurotransmitter-gated ion-channel ligand-binding domain / Neurotransmitter-gated ion-channel transmembrane domain superfamily / Gamma-aminobutyric acid A receptor/Glycine receptor alpha / Neurotransmitter-gated ion-channel transmembrane domain / Neurotransmitter-gated ion-channel / Gamma-aminobutyric-acid A receptor, alpha 1 subunit / Neurotransmitter-gated ion-channel, conserved site / Gamma-aminobutyric-acid A receptor, beta subunit / Neurotransmitter-gated ion-channel ligand-binding domain superfamily / Gamma-aminobutyric-acid A receptor, gamma subunit ...Neurotransmitter-gated ion-channel ligand-binding domain / Neurotransmitter-gated ion-channel transmembrane domain superfamily / Gamma-aminobutyric acid A receptor/Glycine receptor alpha / Neurotransmitter-gated ion-channel transmembrane domain / Neurotransmitter-gated ion-channel / Gamma-aminobutyric-acid A receptor, alpha 1 subunit / Neurotransmitter-gated ion-channel, conserved site / Gamma-aminobutyric-acid A receptor, beta subunit / Neurotransmitter-gated ion-channel ligand-binding domain superfamily / Gamma-aminobutyric-acid A receptor, gamma subunit / Neurotransmitter-gated ion-channel ligand binding domain / Neurotransmitter-gated ion-channel transmembrane region / Gamma-aminobutyric-acid A receptor, alpha subunit / Neurotransmitter-gated ion-channels signature. / GABA A receptor activation / GABA A receptor activation / GABA receptor activation / Gamma-aminobutyric-acid A receptor, gamma 2 subunit / benzodiazepine receptor activity / inner ear receptor cell development / inhibitory extracellular ligand-gated ion channel activity / GABA-gated chloride ion channel activity / GABA-A receptor complex / GABA receptor complex / GABA-A receptor 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 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 / 3.58 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 9, 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,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,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)337,96243
Polyers329,4296
Non-polymers8,53337
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)42130
ΔGint (kcal/M)-90
Surface area (Å2)72830
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,Gamma-aminobutyric acid receptor subunit alpha-1 / GABA(A) receptor subunit alpha-1


Mass: 52916.602 Da / Num. of mol.: 2
Details: Bovine GABRA1 signal peptide: MKKSPGLSDY LWAWTLFLST LTGRSYG FLAG tag: DYKDDDDK,Bovine GABRA1 signal ...Bovine GABRA1 signal peptide: MKKSPGLSDY LWAWTLFLST LTGRSYG FLAG tag: DYKDDDDK,Bovine GABRA1 signal peptide: MKKSPGLSDY LWAWTLFLST LTGRSYG FLAG tag: DYKDDDDK
Source: (gene. exp.) Bos taurus (cattle), (gene. exp.) Homo sapiens (human)
Gene: GABRA1 / Cell line (production host): HEK293S / Production host: Homo sapiens (human) / References: UniProt: P08219, UniProt: P14867
#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 / 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): HEK293S / 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 , 6 types, 37 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.: 12 / Formula: C6H12O6
#9: Chemical ChemComp-ABU / GAMMA-AMINO-BUTANOIC ACID / GAMMA(AMINO)-BUTYRIC ACID


Mass: 103.120 Da / Num. of mol.: 2 / Formula: C4H9NO2
#10: Chemical ChemComp-DZP / 7-CHLORO-1-METHYL-5-PHENYL-1,3-DIHYDRO-2H-1,4-BENZODIAZEPIN-2-ONE


Mass: 284.740 Da / Num. of mol.: 3 / Formula: C16H13ClN2O

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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 diazepam (Valium), GABA and megabody Mb38.COMPLEX1,2,3,40MULTIPLE SOURCES
2heteromeric alpha1beta3gamma2L GABA(A)RCOMPLEX1,2,31RECOMBINANT
3megabody Mb38COMPLEX41RECOMBINANT
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
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: 130000 / Nominal defocus max: 3600 nm / Nominal defocus min: 2400 nm / Cs: 2.7 mm / C2 aperture diameter: 5 microns
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 62 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategoryDetails
1RELION3.0particle selection
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
Particle selectionNumber of particles selected: 233543
SymmetryPoint symmetry: C1
3D reconstructionResolution: 3.58 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 55077 / Symmetry type: POINT
Atomic model buildingRef protocol: FLEXIBLE FIT / Ref space: REAL

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