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- EMDB-0280: CryoEM structure of human full-length alpha1beta3gamma2L GABA(A)R... -

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Basic information

Entry
Database: EMDB / ID: 0280
TitleCryoEM structure of human full-length alpha1beta3gamma2L GABA(A)R in complex with bicuculline and megabody Mb38.
Map dataSynaptic human full-length a1b3g2L GABAAR in complex with bicuculline and megabody Mb38. Sharpened and filtered map (generated with Relion post-processing).
SampleHuman full-length heteromeric alpha1beta3gamma2L GABA(A)R in complex with bicuculline and megabody Mb38.
  • Human full-length heteromeric alpha1beta3gamma2L GABA(A)R
  • megabody Mb38
  • (Gamma-aminobutyric acid receptor subunit ...) x 3
  • Megabody Mb38
  • (ligand) x 5
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
SourceHomo sapiens (human) / Lama glama (llama)
Methodsingle 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 KW / Aricescu AR
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 ReportPDB-ID: 6huk

SummaryFull reportAbout validation report
DateDeposition: Oct 8, 2018 / Header (metadata) release: Nov 7, 2018 / Map release: Jan 2, 2019 / Last update: Jan 16, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.07
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.07
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: : PDB-6huk
  • Surface level: 0.07
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_0280.map.gz (map file in CCP4 format, 44958 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
224 pix
1.07 Å/pix.
= 239.68 Å
224 pix
1.07 Å/pix.
= 239.68 Å
224 pix
1.07 Å/pix.
= 239.68 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density
Contour Level:0.07 (by author), 0.07 (movie #1):
Minimum - Maximum-0.32086563 - 0.4944583
Average (Standard dev.)0.0012150024 (0.015643317)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions224224224
Origin0.00.00.0
Limit223.0223.0223.0
Spacing224224224
CellA=B=C: 239.68001 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z224224224
origin x/y/z0.0000.0000.000
length x/y/z239.680239.680239.680
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS224224224
D min/max/mean-0.3210.4940.001

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Supplemental data

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Sample components

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Entire Human full-length heteromeric alpha1beta3gamma2L GABA(A)R in comp...

EntireName: Human full-length heteromeric alpha1beta3gamma2L GABA(A)R in complex with bicuculline and megabody Mb38.
Number of components: 12

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Component #1: protein, Human full-length heteromeric alpha1beta3gamma2L GABA(A)...

ProteinName: Human full-length heteromeric alpha1beta3gamma2L GABA(A)R in complex with bicuculline and megabody Mb38.
Recombinant expression: No
MassTheoretical: 330 kDa

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Component #2: protein, Human full-length heteromeric alpha1beta3gamma2L GABA(A)R

ProteinName: Human full-length heteromeric alpha1beta3gamma2L GABA(A)R
Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human) / Cell of expression system: HEK293S
Source (natural)Location in cell: Plasma membrane / Organ or tissue: Brain

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Component #3: protein, megabody Mb38

ProteinName: megabody Mb38 / Recombinant expression: No
SourceSpecies: Lama glama (llama)
Source (engineered)Expression System: Esherichia coli

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Component #4: protein, Gamma-aminobutyric acid receptor subunit alpha-1,Gamma-a...

ProteinName: Gamma-aminobutyric acid receptor subunit alpha-1,Gamma-aminobutyric acid receptor subunit alpha-1
Details: Potential signal peptide: MKKSPGLSDY LWAWTLFLST LTGRSYG FLAG tag: DYKDDDDK,Potential signal peptide: MKKSPGLSDY LWAWTLFLST LTGRSYG FLAG tag: DYKDDDDK
Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 52.916602 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human)

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Component #5: protein, Gamma-aminobutyric acid receptor subunit beta-3

ProteinName: Gamma-aminobutyric acid receptor subunit beta-3 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 54.444578 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human)

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Component #6: protein, Gamma-aminobutyric acid receptor subunit gamma-2

ProteinName: Gamma-aminobutyric acid receptor subunit gamma-2 / Details: Linker sequence: GGSGGSGGSGK 1D4 tag: TETSQVAPA / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 56.922055 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human)

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Component #7: protein, Megabody Mb38

ProteinName: Megabody Mb38 / Details: 6His tag: HHHHHH EPEA tag: EPEA / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 57.784301 kDa
SourceSpecies: Lama glama (llama)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #8: ligand, bicuculline methochloride

LigandName: bicuculline methochloride / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 0.367352 kDa

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Component #9: ligand, [(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,...

LigandName: [(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
Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 0.746566 kDa

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Component #10: ligand, N-ACETYL-D-GLUCOSAMINE

LigandName: N-ACETYL-D-GLUCOSAMINEN-Acetylglucosamine / Number of Copies: 14 / Recombinant expression: No
MassTheoretical: 0.221208 kDa

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Component #11: ligand, BETA-D-MANNOSE

LigandName: BETA-D-MANNOSE / Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 0.180156 kDa

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Component #12: ligand, ALPHA-D-MANNOSE

LigandName: ALPHA-D-MANNOSE / Number of Copies: 12 / Recombinant expression: No
MassTheoretical: 0.180156 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.1 mg/ml / pH: 7.6
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 3 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 75000.0 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 500.0 - 700.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: FEI FALCON III (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 30536
3D reconstructionSoftware: RELION / Resolution: 3.69 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Modeling #1Refinement protocol: flexible
Output model

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