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- PDB-7l6u: Unliganded ELIC in POPC-only nanodiscs at 3.3-Angstrom resolution -

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Basic information

Entry
Database: PDB / ID: 7l6u
TitleUnliganded ELIC in POPC-only nanodiscs at 3.3-Angstrom resolution
ComponentsGamma-aminobutyric-acid receptor subunit beta-1
KeywordsMEMBRANE PROTEIN / Pentameric Ligand-gated Ion Channels / Nanodisc / Cys-loop Receptor / Styrene Maleic Acid Copolymer / Protein-lipid Interface
Function / homology
Function and homology information


extracellular ligand-gated monoatomic ion channel activity / transmembrane signaling receptor activity / membrane / identical protein binding
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 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsKumar, P. / Grosman, C.
Funding support United States, 1items
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, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 23, 2021Provider: repository / Type: Initial release
Revision 1.1Jul 14, 2021Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: Gamma-aminobutyric-acid receptor subunit beta-1
B: Gamma-aminobutyric-acid receptor subunit beta-1
C: Gamma-aminobutyric-acid receptor subunit beta-1
D: Gamma-aminobutyric-acid receptor subunit beta-1
E: Gamma-aminobutyric-acid receptor subunit beta-1


Theoretical massNumber of molelcules
Total (without water)184,3955
Polymers184,3955
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 36879.000 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Dickeya dadantii (strain 3937) (bacteria)
Strain: 3937 / Gene: Dda3937_00520
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: E0SJQ4

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Erwinia chrysanthemi ligand gated ion channel in lipid nanodiscs
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 184.2 kDa/nm / Experimental value: NO
Source (natural)Organism: Dickeya dadantii 3937 (bacteria)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 8 / Details: 150 mM NaCl and 10 mM sodium phosphate, pH 8.0.
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMSodium phosphate bufferNa2HPO41
2Sodium phosphate bufferNaH2PO41
3150 mMSodium chlorideNaClSodium chloride1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: Homemade
VitrificationInstrument: SPOTITON / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Image recordingElectron dose: 63.56 e/Å2 / Detector mode: COUNTING / Film or detector model: DIRECT ELECTRON DE-16 (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM softwareName: cryoSPARC / Version: v2.15 / Category: 3D reconstruction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 138410
SymmetryPoint symmetry: C5 (5 fold cyclic)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 38381 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00913015
ELECTRON MICROSCOPYf_angle_d0.72917735
ELECTRON MICROSCOPYf_dihedral_angle_d11.1481710
ELECTRON MICROSCOPYf_chiral_restr0.0521975
ELECTRON MICROSCOPYf_plane_restr0.0052260

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