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- EMDB-17960: Structure of GABAAR determined by cryoEM at 100 keV -

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Basic information

Entry
Database: EMDB / ID: EMD-17960
TitleStructure of GABAAR determined by cryoEM at 100 keV
Map data
Sample
  • Complex: Human GABA(A)R-beta3 homopentamer in complex with Megabody-25 in lipid nanodisc
    • Protein or peptide: Gamma-aminobutyric acid receptor subunit beta-3
    • Protein or peptide: Megabody Mb25
  • Ligand: HEXADECANE
  • Ligand: DECANE
  • Ligand: HISTAMINE
  • Ligand: CHLORIDE IONChloride
  • Ligand: Etomidate
KeywordsPentameric ligand-gated ion channel / Neurotrasmitter receptor / GABA(A) receptor / MEMBRANE PROTEIN
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: 3.6 Å
AuthorsMcMullan G / Naydenova K / Mihaylov D / Peet MJ / Wilson H / Yamashita K / Dickerson JL / Chen S / Cannone G / Lee Y ...McMullan G / Naydenova K / Mihaylov D / Peet MJ / Wilson H / Yamashita K / Dickerson JL / Chen S / Cannone G / Lee Y / Hutchings KA / Gittins O / Sobhy M / Wells T / El-Gomati MM / Dalby J / Meffert M / Schulze-Briese C / Henderson R / Russo CJ
Funding support United Kingdom, 6 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MC UP 120117 United Kingdom
Medical Research Council (MRC, United Kingdom)MC U105184322 United Kingdom
Wellcome Trust220526/B/20/Z United Kingdom
Engineering and Physical Sciences Research CouncilR122522 United Kingdom
Innovate UK103806 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/T003677/1 United Kingdom
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Structure determination by cryoEM at 100 keV.
Authors: Greg McMullan / Katerina Naydenova / Daniel Mihaylov / Keitaro Yamashita / Mathew J Peet / Hugh Wilson / Joshua L Dickerson / Shaoxia Chen / Giuseppe Cannone / Yang Lee / Katherine A ...Authors: Greg McMullan / Katerina Naydenova / Daniel Mihaylov / Keitaro Yamashita / Mathew J Peet / Hugh Wilson / Joshua L Dickerson / Shaoxia Chen / Giuseppe Cannone / Yang Lee / Katherine A Hutchings / Olivia Gittins / Mohamed A Sobhy / Torquil Wells / Mohamed M El-Gomati / Jason Dalby / Matthias Meffert / Clemens Schulze-Briese / Richard Henderson / Christopher J Russo /
Abstract: Electron cryomicroscopy can, in principle, determine the structures of most biological molecules but is currently limited by access, specimen preparation difficulties, and cost. We describe a purpose- ...Electron cryomicroscopy can, in principle, determine the structures of most biological molecules but is currently limited by access, specimen preparation difficulties, and cost. We describe a purpose-built instrument operating at 100 keV-including advances in electron optics, detection, and processing-that makes structure determination fast and simple at a fraction of current costs. The instrument attains its theoretical performance limits, allowing atomic resolution imaging of gold test specimens and biological molecular structure determination in hours. We demonstrate its capabilities by determining the structures of eleven different specimens, ranging in size from 140 kDa to 2 MDa, using a fraction of the data normally required. CryoEM with a microscope designed specifically for high-efficiency, on-the-spot imaging of biological molecules will expand structural biology to a wide range of previously intractable problems.
History
DepositionJul 17, 2023-
Header (metadata) releaseNov 29, 2023-
Map releaseNov 29, 2023-
UpdateJan 31, 2024-
Current statusJan 31, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17960.png

Downloads & links

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Map

FileDownload / File: emd_17960.map.gz / Format: CCP4 / Size: 120.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.828 Å
Density
Contour LevelBy AUTHOR: 0.04
Minimum - Maximum-0.1014589 - 0.14704965
Average (Standard dev.)0.00021151654 (±0.004198078)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions316316316
Spacing316316316
CellA=B=C: 261.648 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17960_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_17960_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_17960_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
    • Protein or peptide: Gamma-aminobutyric acid receptor subunit beta-3
    • Protein or peptide: Megabody Mb25
  • Ligand: HEXADECANE
  • Ligand: DECANE
  • Ligand: HISTAMINE
  • Ligand: CHLORIDE IONChloride
  • Ligand: Etomidate

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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
Source (natural)Organism: Homo sapiens (human)

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

MacromoleculeName: 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

UniProtKB: Gamma-aminobutyric acid receptor subunit beta-3

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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: HISTAMINE

MacromoleculeName: HISTAMINE / type: ligand / ID: 7 / 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 #8: CHLORIDE ION

MacromoleculeName: CHLORIDE ION / type: ligand / ID: 8 / Number of copies: 2 / Formula: CL
Molecular weightTheoretical: 35.453 Da

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Macromolecule #9: Etomidate

MacromoleculeName: Etomidate / type: ligand / ID: 9 / Number of copies: 1 / Formula: V8D
Molecular weightTheoretical: 244.289 Da
Chemical component information

ChemComp-V8D:
Etomidate / Etomidate

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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: 7.4
GridModel: UltrAuFoil R0./1 / Material: GOLD
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeJEOL 1400/HR + YPS FEG
Electron beamAcceleration voltage: 100 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: DECTRIS SINGLA (1k x 1k) / Digitization - Dimensions - Width: 1030 pixel / Digitization - Dimensions - Height: 1066 pixel / Average electron dose: 51.0 e/Å2

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Image processing

Startup modelType of model: EMDB MAP
EMDB ID:

13256

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C5 (5 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 23477
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7a5v


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: RECIPROCAL
Output model

PDB-8pvb:
Structure of GABAAR determined by cryoEM at 100 keV

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