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

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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
Sample
  • Complex: Human GABA(A)R-beta3 homopentamer in complex with Megabody-25 in lipid nanodisc
    • Complex: Human GABA(A)R-beta3 homopentamer
      • Protein or peptide: Gamma-aminobutyric acid receptor subunit beta-3,Gamma-aminobutyric acid receptor subunit beta-3
    • Complex: Megabody-25
      • Protein or peptide: Megabody Mb25
  • Ligand: HEXADECANE
  • Ligand: DECANE
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: N-OCTANEOctane
  • Ligand: HISTAMINE
  • Ligand: CHLORIDE IONChloride
  • Ligand: water
Function / homology
Function and homology information


GABA receptor activation / GABA-gated chloride ion channel activity / cellular response to histamine / inhibitory synapse assembly / GABA-A receptor activity / GABA-A receptor complex / neurotransmitter receptor activity / gamma-aminobutyric acid signaling pathway / synaptic transmission, GABAergic / roof of mouth development ...GABA receptor activation / GABA-gated chloride ion channel activity / cellular response to histamine / inhibitory synapse assembly / GABA-A receptor activity / GABA-A receptor complex / neurotransmitter receptor activity / gamma-aminobutyric acid signaling pathway / synaptic transmission, GABAergic / roof of mouth development / Signaling by ERBB4 / chloride channel complex / chloride transmembrane transport / cytoplasmic vesicle membrane / postsynaptic membrane / neuron projection / synapse / signal transduction / identical protein binding / plasma membrane
Similarity search - Function
Gamma-aminobutyric-acid A receptor, beta subunit / Gamma-aminobutyric acid A receptor/Glycine receptor alpha / Neurotransmitter-gated ion-channel, conserved site / Neurotransmitter-gated ion-channels signature. / Neurotransmitter-gated ion-channel transmembrane domain / Neurotransmitter-gated ion-channel transmembrane region / Neurotransmitter-gated ion-channel transmembrane domain superfamily / Neuronal acetylcholine receptor / Neurotransmitter-gated ion-channel / Neurotransmitter-gated ion-channel ligand-binding domain ...Gamma-aminobutyric-acid A receptor, beta subunit / Gamma-aminobutyric acid A receptor/Glycine receptor alpha / Neurotransmitter-gated ion-channel, conserved site / Neurotransmitter-gated ion-channels signature. / Neurotransmitter-gated ion-channel transmembrane domain / Neurotransmitter-gated ion-channel transmembrane region / Neurotransmitter-gated ion-channel transmembrane domain superfamily / Neuronal acetylcholine receptor / Neurotransmitter-gated ion-channel / Neurotransmitter-gated ion-channel ligand-binding domain / Neurotransmitter-gated ion-channel ligand-binding domain superfamily / Neurotransmitter-gated ion-channel ligand binding domain
Similarity search - Domain/homology
Gamma-aminobutyric acid receptor subunit beta-3
Similarity search - Component
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.
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
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  • 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
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  • 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

emd_11657.png

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)
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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Mask #1

Fileemd_11657_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_11657_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_11657_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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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
Components
  • Complex: Human GABA(A)R-beta3 homopentamer in complex with Megabody-25 in lipid nanodisc
    • Complex: Human GABA(A)R-beta3 homopentamer
      • Protein or peptide: Gamma-aminobutyric acid receptor subunit beta-3,Gamma-aminobutyric acid receptor subunit beta-3
    • Complex: Megabody-25
      • Protein or peptide: Megabody Mb25
  • Ligand: HEXADECANE
  • Ligand: DECANE
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: N-OCTANEOctane
  • Ligand: HISTAMINE
  • Ligand: CHLORIDE IONChloride
  • Ligand: water

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Supramolecule #1: Human GABA(A)R-beta3 homopentamer in complex with Megabody-25 in ...

SupramoleculeName: Human GABA(A)R-beta3 homopentamer in complex with Megabody-25 in lipid nanodisc
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Molecular weightTheoretical: 485 KDa

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Supramolecule #2: Human GABA(A)R-beta3 homopentamer

SupramoleculeName: Human GABA(A)R-beta3 homopentamer / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant strain: HEK293S

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Supramolecule #3: Megabody-25

SupramoleculeName: Megabody-25 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Lama glama (llama)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: Gamma-aminobutyric acid receptor subunit beta-3,Gamma-aminobutyri...

MacromoleculeName: Gamma-aminobutyric acid receptor subunit beta-3,Gamma-aminobutyric acid receptor subunit beta-3
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 45.744285 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: EMDEKTTGWR GGHVVEGLAG ELEQLRARLE HHPQGQREPD YDIPTTENLY FQGTGQSVND PGNMSFVKET VDKLLKGYDI RLRPDFGGP PVCVGMNIDI ASIDMVSEVN MDYTLTMYFQ QYWRDKRLAY SGIPLNLTLD NRVADQLWVP DTYFLNDKKS F VHGVTVKN ...String:
EMDEKTTGWR GGHVVEGLAG ELEQLRARLE HHPQGQREPD YDIPTTENLY FQGTGQSVND PGNMSFVKET VDKLLKGYDI RLRPDFGGP PVCVGMNIDI ASIDMVSEVN MDYTLTMYFQ QYWRDKRLAY SGIPLNLTLD NRVADQLWVP DTYFLNDKKS F VHGVTVKN RMIRLHPDGT VLYGLRITTT AACMMDLRRY PLDEQNCTLE IESYGYTTDD IEFYWRGGDK AVTGVERIEL PQ FSIVEHR LVSRNVVFAT GAYPRLSLSF RLKRNIGYFI LQTYMPSILI TILSWVSFWI NYDASAARVA LGITTVLTMT TIN THLRET LPKIPYVTAI DMYLMGCFVF VFLALLEYAF VNYIFFSQPA RAAAIDRWSR IVFPFTFSLF NLVYWLYYVN

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Macromolecule #2: Megabody Mb25

MacromoleculeName: Megabody Mb25 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 56.300629 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: QVQLVESGGG LVQTKTTTSV IDTTNDAQNL LTQAQTIVNT LKDYCPILIA KSSSSNGGTN NANTPSWQTA GGGKNSCATF GAEFSAASD MINNAQKIVQ ETQQLSANQP KNITQPHNLN LNSPSSLTAL AQKMLKNAQS QAEILKLANQ VESDFNKLSS G HLKDYIGK ...String:
QVQLVESGGG LVQTKTTTSV IDTTNDAQNL LTQAQTIVNT LKDYCPILIA KSSSSNGGTN NANTPSWQTA GGGKNSCATF GAEFSAASD MINNAQKIVQ ETQQLSANQP KNITQPHNLN LNSPSSLTAL AQKMLKNAQS QAEILKLANQ VESDFNKLSS G HLKDYIGK CDASAISSAN MTMQNQKNNW GNGCAGVEET QSLLKTSAAD FNNQTPQINQ AQNLANTLIQ ELGNNTYEQL SR LLTNDNG TNSKTSAQAI NQAVNNLNER AKTLAGGTTN SPAYQATLLA LRSVLGLWNS MGYAVICGGY TKSPGENNQK DFH YTDENG NGTTINCGGS TNSNGTHSYN GTNTLKADKN VSLSIEQYEK IHEAYQILSK ALKQAGLAPL NSKGEKLEAH VTTS KYGSL RLSCAASGHT FNYPIMGWFR QAPGKEREFV GAISWSGGST SYADSVKDRF TISRDNAKNT VYLEMNNLKP EDTAV YYCA AKGRYSGGLY YPTNYDYWGQ GTQVTVSSHH HHHHEPEA

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Macromolecule #5: HEXADECANE

MacromoleculeName: HEXADECANE / type: ligand / ID: 5 / Number of copies: 1 / Formula: R16
Molecular weightTheoretical: 226.441 Da
Chemical component information

ChemComp-R16:
HEXADECANE / Hexadecane

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Macromolecule #6: DECANE

MacromoleculeName: DECANE / type: ligand / ID: 6 / Number of copies: 4 / Formula: D10
Molecular weightTheoretical: 142.282 Da
Chemical component information

ChemComp-D10:
DECANE / Decane

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Macromolecule #7: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 7 / Number of copies: 1 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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Macromolecule #8: N-OCTANE

MacromoleculeName: N-OCTANE / type: ligand / ID: 8 / Number of copies: 1 / Formula: OCT
Molecular weightTheoretical: 114.229 Da
Chemical component information

ChemComp-OCT:
N-OCTANE / Octane

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Macromolecule #9: HISTAMINE

MacromoleculeName: HISTAMINE / type: ligand / ID: 9 / Number of copies: 1 / Formula: HSM
Molecular weightTheoretical: 111.145 Da
Chemical component information

ChemComp-HSM:
HISTAMINE / neurotransmitter, hormone*YM / Histamine

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Macromolecule #10: CHLORIDE ION

MacromoleculeName: CHLORIDE ION / type: ligand / ID: 10 / Number of copies: 2 / Formula: CL
Molecular weightTheoretical: 35.453 Da

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Macromolecule #11: water

MacromoleculeName: water / type: ligand / ID: 11 / Number of copies: 203 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.6
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 287 K / Instrument: LEICA PLUNGER

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.1 µm / Nominal defocus min: 0.3 µm / Nominal magnification: 165000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4.1.13)
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationSoftware - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionApplied symmetry - Point group: C5 (5 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 1.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 371693
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: AB INITIO MODEL
Output model

PDB-7a5v:
CryoEM structure of a human gamma-aminobutyric acid receptor, the GABA(A)R-beta3 homopentamer, in complex with histamine and megabody Mb25 in lipid nanodisc

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