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- EMDB-23208: Unliganded ELIC in POPC-only nanodiscs at 3.3-Angstrom resolution -

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Basic information

Entry
Database: EMDB / ID: EMD-23208
TitleUnliganded ELIC in POPC-only nanodiscs at 3.3-Angstrom resolution
Map dataUnliganded ELIC in POPC-only nanodiscs at 3.3-Angstrom resolution
Sample
  • Complex: Erwinia chrysanthemi ligand gated ion channel in lipid nanodiscs
    • Protein or peptide: Gamma-aminobutyric-acid receptor subunit beta-1
KeywordsPentameric Ligand-gated Ion Channels / Nanodisc / Cys-loop Receptor / Styrene Maleic Acid Copolymer / Membrane Protein / Protein-lipid Interface
Function / homology
Function and homology information


extracellular ligand-gated monoatomic ion channel activity / regulation of membrane potential / transmembrane signaling receptor activity / neuron projection / signal transduction / identical protein binding / membrane
Similarity search - Function
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-1
Similarity search - Component
Biological speciesDickeya dadantii 3937 (bacteria) / Dickeya dadantii (strain 3937) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsKumar P / Grosman C
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS042169 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2021
Title: Structure and function at the lipid-protein interface of a pentameric ligand-gated ion channel.
Authors: Pramod Kumar / Gisela D Cymes / Claudio Grosman /
Abstract: Although it has long been proposed that membrane proteins may contain tightly bound lipids, their identity, the structure of their binding sites, and their functional and structural relevance have ...Although it has long been proposed that membrane proteins may contain tightly bound lipids, their identity, the structure of their binding sites, and their functional and structural relevance have remained elusive. To some extent, this is because tightly bound lipids are often located at the periphery of proteins, where the quality of density maps is usually poorer, and because they may be outcompeted by detergent molecules used during standard purification procedures. As a step toward characterizing natively bound lipids in the superfamily of pentameric ligand-gated ion channels (pLGICs), we applied single-particle cryogenic electron microscopy to fragments of native membrane obtained in the complete absence of detergent-solubilization steps. Because of the heterogeneous lipid composition of membranes in the secretory pathway of eukaryotic cells, we chose to study a bacterial pLGIC (ELIC) expressed in 's inner membrane. We obtained a three-dimensional reconstruction of unliganded ELIC (2.5-Å resolution) that shows clear evidence for two types of tightly bound lipid at the protein-bulk-membrane interface. One of them was consistent with a "regular" diacylated phospholipid, in the cytoplasmic leaflet, whereas the other one was consistent with the tetra-acylated structure of cardiolipin, in the periplasmic leaflet. Upon reconstitution in polar-lipid bilayers, ELIC retained the functional properties characteristic of members of this superfamily, and thus, the fitted atomic model is expected to represent the (long-debated) unliganded-closed, "resting" conformation of this ion channel. Notably, the addition of cardiolipin to phosphatidylcholine membranes restored the ion-channel activity that is largely lost in phosphatidylcholine-only bilayers.
History
DepositionDec 23, 2020-
Header (metadata) releaseJun 23, 2021-
Map releaseJun 23, 2021-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.17
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.17
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7l6u
  • Surface level: 0.17
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23208.png

Downloads & links

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Map

FileDownload / File: emd_23208.map.gz / Format: CCP4 / Size: 31.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnliganded ELIC in POPC-only nanodiscs at 3.3-Angstrom resolution
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 202 pix.
= 221.392 Å		1.1 Å/pix.
x 202 pix.
= 221.392 Å		1.1 Å/pix.
x 202 pix.
= 221.392 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.096 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.17 / Movie #1: 0.17
Minimum - Maximum-1.2697724 - 1.8350552
Average (Standard dev.)0.00290594 (±0.06405737)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions202202202
Spacing202202202
CellA=B=C: 221.392 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0961.0961.096
M x/y/z202202202
origin x/y/z0.0000.0000.000
length x/y/z221.392221.392221.392
α/β/γ90.00090.00090.000
start NX/NY/NZ192139186
NX/NY/NZ211274246
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS202202202
D min/max/mean-1.2701.8350.003

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Supplemental data

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Mask #1

Fileemd_23208_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 1

Fileemd_23208_half_map_1.map
AnnotationHalf-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 2

Fileemd_23208_half_map_2.map
AnnotationHalf-map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

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Sample components

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Entire : Erwinia chrysanthemi ligand gated ion channel in lipid nanodiscs

EntireName: Erwinia chrysanthemi ligand gated ion channel in lipid nanodiscs
Components
  • Complex: Erwinia chrysanthemi ligand gated ion channel in lipid nanodiscs
    • Protein or peptide: Gamma-aminobutyric-acid receptor subunit beta-1

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Supramolecule #1: Erwinia chrysanthemi ligand gated ion channel in lipid nanodiscs

SupramoleculeName: Erwinia chrysanthemi ligand gated ion channel in lipid nanodiscs
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Dickeya dadantii 3937 (bacteria)
Molecular weightTheoretical: 184.2 kDa/nm

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

MacromoleculeName: Gamma-aminobutyric-acid receptor subunit beta-1 / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Dickeya dadantii (strain 3937) (bacteria) / Strain: 3937
Molecular weightTheoretical: 36.879 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: APADNAADAR PVDVSVSIFI NKIYGVNTLE QTYKVDGYIV AQWTGKPRKT PGDKPLIVEN TQIERWINNG LWVPALEFIN VVGSPDTGN KRLMLFPDGR VIYNARFLGS FSNDMDFRLF PFDRQQFVLE LEPFSYNNQQ LRFSDIQVYT ENIDNEEIDE W WIRGKAST ...String:
APADNAADAR PVDVSVSIFI NKIYGVNTLE QTYKVDGYIV AQWTGKPRKT PGDKPLIVEN TQIERWINNG LWVPALEFIN VVGSPDTGN KRLMLFPDGR VIYNARFLGS FSNDMDFRLF PFDRQQFVLE LEPFSYNNQQ LRFSDIQVYT ENIDNEEIDE W WIRGKAST HISDIRYDHL SSVQPNQNEF SRITVRIDAV RNPSYYLWSF ILPLGLIIAA SWSVFWLESF SERLQTSFTL ML TVVAYAF YTSNILPRLP YTTVIDQMII AGYGSIFAAI LLIIFAHHRQ ANGVEDDLLI QRCRLAFPLG FLAIGCVLVI RGI TL

UniProtKB: Gamma-aminobutyric-acid receptor subunit beta-1

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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

Concentration2 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
10.0 mMNa2HPO4Sodium phosphate buffer
NaH2PO4Sodium phosphate buffer
150.0 mMNaClSodium chloride

Details: 150 mM NaCl and 10 mM sodium phosphate, pH 8.0.
GridModel: Homemade / Material: GOLD
VitrificationCryogen name: ETHANE / Instrument: SPOTITON

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: DIRECT ELECTRON DE-16 (4k x 4k) / Detector mode: COUNTING / Average electron dose: 63.56 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 138410
Startup modelType of model: PDB ENTRY
Final reconstructionApplied symmetry - Point group: C5 (5 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v2.15) / Number images used: 38381
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE

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