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

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Basic information

Entry
Database: PDB / ID: 6huk
TitleCryoEM structure of human full-length alpha1beta3gamma2L GABA(A)R in complex with bicuculline and megabody Mb38.
Components
  • (Gamma-aminobutyric acid receptor subunit ...) x 3
  • Megabody Mb38
KeywordsMEMBRANE PROTEIN / GABAAR / Membrane / Channel / Nanobody / Megabody / Cys-loop / PLGIC / Inhibition / Signalling / CNS / Neurons / Chloride / Ion / GABA / BCC / Bicuculline / Antagonist
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 / 3.69 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,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)337,63640
Polyers329,4296
Non-polymers8,20734
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)41150
ΔGint (kcal/M)-96
Surface area (Å2)73010
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: Potential signal peptide: MKKSPGLSDY LWAWTLFLST LTGRSYG FLAG tag: DYKDDDDK,Potential signal peptide: ...Potential signal peptide: MKKSPGLSDY LWAWTLFLST LTGRSYG FLAG tag: DYKDDDDK,Potential 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 / Cell line (production host): HEK293S / 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 , 5 types, 34 molecules

#5: Chemical ChemComp-H0Z / bicuculline methochloride


Mass: 367.352 Da / Num. of mol.: 2 / Formula: C20H17NO6
#6: 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
#7: Chemical
ChemComp-NAG / N-ACETYL-D-GLUCOSAMINE


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


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


Mass: 180.156 Da / Num. of mol.: 12 / Formula: C6H12O6

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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 bicuculline and megabody Mb38.COMPLEX1, 2, 3, 40MULTIPLE SOURCES
2Human full-length heteromeric 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)
33562Esherichia 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 supportGrid material: GOLD / 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
9PHENIXmodel refinement
10RELION3.0initial Euler assignmentRefine3D
11RELION3.0final Euler assignmentRefine3D
12RELION3.0classificationClass3D
13RELION3.03D reconstructionRefine3D
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNumber of particles selected: 489434
SymmetryPoint symmetry: C1
3D reconstructionResolution: 3.69 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 30536 / Symmetry type: POINT
Atomic model buildingRef protocol: FLEXIBLE FIT

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