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- EMDB-11657: CryoEM structure of a human gamma-aminobutyric acid receptor, the... -

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Basic information

Entry
Database: EMDB / ID: EMD-11657
TitleCryoEM structure of a human gamma-aminobutyric acid receptor, the GABA(A)R-beta3 homopentamer, in complex with histamine and megabody Mb25 in lipid nanodisc
Map data
SampleHuman GABA(A)R-beta3 homopentamer in complex with Megabody-25 in lipid nanodisc
  • Human GABA(A)R-beta3 homopentamer
  • Megabody-25
  • Gamma-aminobutyric acid receptor subunit beta-3,Gamma-aminobutyric acid receptor subunit beta-3
  • Megabody Mb25
  • (ligand) x 7
Function / homology
Function and homology information


inner ear receptor cell development / GABA-A receptor activity / GABA-A receptor complex / GABA-gated chloride ion channel activity / cellular response to histamine / inhibitory synapse assembly / gamma-aminobutyric acid signaling pathway / innervation / inhibitory postsynaptic potential / neurotransmitter receptor activity ...inner ear receptor cell development / GABA-A receptor activity / GABA-A receptor complex / GABA-gated chloride ion channel activity / cellular response to histamine / inhibitory synapse assembly / gamma-aminobutyric acid signaling pathway / innervation / inhibitory postsynaptic potential / neurotransmitter receptor activity / nervous system process / GABA-ergic synapse / chloride channel complex / cochlea development / chloride transmembrane transport / roof of mouth development / transmitter-gated ion channel activity involved in regulation of postsynaptic membrane potential / ion transmembrane transport / regulation of membrane potential / sensory perception of sound / chemical synaptic transmission / cytoplasmic vesicle membrane / postsynaptic membrane / negative regulation of neuron apoptotic process / neuron projection / synapse / signal transduction / integral component of plasma membrane / identical protein binding / plasma membrane
Neurotransmitter-gated ion-channel ligand-binding domain superfamily / Neurotransmitter-gated ion-channel transmembrane domain superfamily / Neurotransmitter-gated ion-channel, conserved site / Neurotransmitter-gated ion-channel ligand-binding domain / Neurotransmitter-gated ion-channel / Neurotransmitter-gated ion-channel transmembrane domain / Gamma-aminobutyric acid A receptor/Glycine receptor alpha / Gamma-aminobutyric-acid A receptor, beta subunit
Gamma-aminobutyric acid receptor subunit beta-3
Biological speciesHomo sapiens (human) / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 1.7 Å
AuthorsNakane T / Kotecha A / Sente A / Yamashita K / McMullan G / Masiulis S / Brown PMGE / Grigoras IT / Malinauskaite L / Malinauskas T ...Nakane T / Kotecha A / Sente A / Yamashita K / McMullan G / Masiulis S / Brown PMGE / Grigoras IT / Malinauskaite L / Malinauskas T / Miehling J / Yu L / Karia D / Pechnikova EV / de Jong E / Keizer J / Bischoff M / McCormack J / Tiemeijer P / Hardwick SW / Chirgadze DY / Murshudov G / Aricescu AR / Scheres SHW
Funding support United Kingdom, 6 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MC_UP_A025_1012 United Kingdom
Medical Research Council (MRC, United Kingdom)MC_UP_1201/15 United Kingdom
Medical Research Council (MRC, United Kingdom)MR/L009609/1 United Kingdom
Wellcome Trust206171/Z/17/Z United Kingdom
Medical Research Council (MRC, United Kingdom)MC_UP_A025_1013 United Kingdom
Wellcome Trust202905/Z/16/Z) United Kingdom
CitationJournal: Nature / Year: 2020
Title: Single-particle cryo-EM at atomic resolution.
Authors: Takanori Nakane / Abhay Kotecha / Andrija Sente / Greg McMullan / Simonas Masiulis / Patricia M G E Brown / Ioana T Grigoras / Lina Malinauskaite / Tomas Malinauskas / Jonas Miehling / ...Authors: Takanori Nakane / Abhay Kotecha / Andrija Sente / Greg McMullan / Simonas Masiulis / Patricia M G E Brown / Ioana T Grigoras / Lina Malinauskaite / Tomas Malinauskas / Jonas Miehling / Tomasz Uchański / Lingbo Yu / Dimple Karia / Evgeniya V Pechnikova / Erwin de Jong / Jeroen Keizer / Maarten Bischoff / Jamie McCormack / Peter Tiemeijer / Steven W Hardwick / Dimitri Y Chirgadze / Garib Murshudov / A Radu Aricescu / Sjors H W Scheres /
Abstract: The three-dimensional positions of atoms in protein molecules define their structure and their roles in biological processes. The more precisely atomic coordinates are determined, the more chemical ...The three-dimensional positions of atoms in protein molecules define their structure and their roles in biological processes. The more precisely atomic coordinates are determined, the more chemical information can be derived and the more mechanistic insights into protein function may be inferred. Electron cryo-microscopy (cryo-EM) single-particle analysis has yielded protein structures with increasing levels of detail in recent years. However, it has proved difficult to obtain cryo-EM reconstructions with sufficient resolution to visualize individual atoms in proteins. Here we use a new electron source, energy filter and camera to obtain a 1.7 Å resolution cryo-EM reconstruction for a human membrane protein, the β3 GABA receptor homopentamer. Such maps allow a detailed understanding of small-molecule coordination, visualization of solvent molecules and alternative conformations for multiple amino acids, and unambiguous building of ordered acidic side chains and glycans. Applied to mouse apoferritin, our strategy led to a 1.22 Å resolution reconstruction that offers a genuine atomic-resolution view of a protein molecule using single-particle cryo-EM. Moreover, the scattering potential from many hydrogen atoms can be visualized in difference maps, allowing a direct analysis of hydrogen-bonding networks. Our technological advances, combined with further approaches to accelerate data acquisition and improve sample quality, provide a route towards routine application of cryo-EM in high-throughput screening of small molecule modulators and structure-based drug discovery.
Validation ReportPDB-ID: 7a5v

SummaryFull reportAbout validation report
History
DepositionAug 22, 2020-
Header (metadata) releaseNov 18, 2020-
Map releaseNov 18, 2020-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7a5v
  • Surface level: 0.025
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7a5v
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_11657.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.81 Å/pix.
x 360 pix.
= 292.212 Å
0.81 Å/pix.
x 360 pix.
= 292.212 Å
0.81 Å/pix.
x 360 pix.
= 292.212 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8117 Å
Density
Contour LevelBy AUTHOR: 0.025 / Movie #1: 0.04
Minimum - Maximum-0.18087125 - 0.38283664
Average (Standard dev.)0.00006339444 (±0.0071915314)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 292.212 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.81170.81170.8117
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z292.212292.212292.212
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-0.1810.3830.000

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Supplemental data

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Segmentation: #1

Fileemd_11657_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: #2

Fileemd_11657_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: #1

Fileemd_11657_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire Human GABA(A)R-beta3 homopentamer in complex with Megabody-25 in ...

EntireName: Human GABA(A)R-beta3 homopentamer in complex with Megabody-25 in lipid nanodisc
Number of components: 12

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Component #1: protein, Human GABA(A)R-beta3 homopentamer in complex with Megabo...

ProteinName: Human GABA(A)R-beta3 homopentamer in complex with Megabody-25 in lipid nanodisc
Recombinant expression: No
MassTheoretical: 485 kDa

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Component #2: protein, Human GABA(A)R-beta3 homopentamer

ProteinName: Human GABA(A)R-beta3 homopentamer / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human) / Strain: HEK293S

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Component #3: protein, Megabody-25

ProteinName: Megabody-25 / Recombinant expression: No
SourceSpecies: Lama glama (llama)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #4: protein, Gamma-aminobutyric acid receptor subunit beta-3,Gamma-am...

ProteinName: Gamma-aminobutyric acid receptor subunit beta-3,Gamma-aminobutyric acid receptor subunit beta-3
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 45.744285 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human)

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Component #5: protein, Megabody Mb25

ProteinName: Megabody Mb25 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 56.300629 kDa
SourceSpecies: Lama glama (llama)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #6: ligand, HEXADECANE

LigandName: HEXADECANE / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.226441 kDa

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Component #7: ligand, DECANE

LigandName: DECANE / Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 0.142282 kDa

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Component #8: ligand, 2-acetamido-2-deoxy-beta-D-glucopyranose

LigandName: 2-acetamido-2-deoxy-beta-D-glucopyranose / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.221208 kDa

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Component #9: ligand, N-OCTANE

LigandName: N-OCTANEOctane / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.114229 kDa

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Component #10: ligand, HISTAMINE

LigandName: HISTAMINE / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.111145 kDa

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Component #11: ligand, CHLORIDE ION

LigandName: CHLORIDE IONChloride / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 3.545305 MDa

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Component #12: ligand, water

LigandName: water / Number of Copies: 203 / Recombinant expression: No
MassTheoretical: 1.801505 MDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionpH: 7.6
VitrificationInstrument: LEICA PLUNGER / Cryogen name: ETHANE / Temperature: 287 K / Humidity: 95 %

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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: 40 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 165000 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 300.0 - 1100.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: OTHER

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C5 (5 fold cyclic) / Number of projections: 371693
3D reconstructionAlgorithm: FOURIER SPACE / Software: RELION / Resolution: 1.7 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement space: RECIPROCAL
Output model

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