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- PDB-7l6q: Unliganded ELIC in styrene-maleic-acid nanodiscs at 2.5-Angstrom ... -

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Basic information

Entry
Database: PDB / ID: 7l6q
TitleUnliganded ELIC in styrene-maleic-acid nanodiscs at 2.5-Angstrom resolution
ComponentsGamma-aminobutyric-acid receptor subunit beta-1
KeywordsMEMBRANE PROTEIN / Pentameric Ligand-gated Ion Channels / Nanodisc / Cys-loop receptor / Styrene-maleic acid / Protein-lipid interface
Function / homology
Function and homology information


extracellular ligand-gated monoatomic ion channel activity / transmembrane signaling receptor activity / 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
CARDIOLIPIN / Chem-PGW / Gamma-aminobutyric-acid receptor subunit beta-1
Similarity search - Component
Biological speciesDickeya dadantii (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsGrosman, C. / Kumar, P.
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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)195,46015
Polymers184,3955
Non-polymers11,06510
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "A"
d_2ens_1chain "B"
d_3ens_1chain "C"
d_4ens_1chain "D"
d_5ens_1chain "E"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDEnd label seq-ID
d_11ens_1CDLCDLA
d_12ens_1PGWTHRB313
d_21ens_1CDLCDLC
d_22ens_1PGWTHRD313
d_31ens_1CDLCDLE
d_32ens_1PGWTHRF313
d_41ens_1CDLCDLG
d_42ens_1PGWTHRH313
d_51ens_1CDLCDLI
d_52ens_1PGWTHRJ313

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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 (E. coli) / Strain (production host): BL 21 / References: UniProt: E0SJQ4
#2: Chemical
ChemComp-PGW / (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]oxy}-1-[(hexadecanoyloxy)methyl]ethyl (9Z)-octadec-9-enoate / 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-[Phospho-(1-glycerol)] / PHOSPHATIDYLGLYCEROL


Mass: 749.007 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C40H77O10P / Comment: phospholipid*YM
#3: Chemical
ChemComp-CDL / CARDIOLIPIN / DIPHOSPHATIDYL GLYCEROL / BIS-(1,2-DIACYL-SN-GLYCERO-3-PHOSPHO)-1',3'-SN-GLYCEROL


Mass: 1464.043 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C81H156O17P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
Has ligand of interestY

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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: Unliganded Erwinia chrysanthemi ligand gated ion channel in styrene-maleic-acid nanodiscs
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 184.2 kDa/nm / Experimental value: NO
Source (natural)Organism: Dickeya dadantii (bacteria)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Strain: BL21
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris(Hydroxymethy)aminomethaneC4H11O3N1
2150 MmSodium ChlorideNaCl1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD
VitrificationInstrument: LEICA EM GP / 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 FIELD
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 56.23 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
CTF correctionType: NONE
SymmetryPoint symmetry: C5 (5 fold cyclic)
3D reconstructionResolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1239532 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 61.5 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00913660
ELECTRON MICROSCOPYf_angle_d0.62318470
ELECTRON MICROSCOPYf_dihedral_angle_d16.0375110
ELECTRON MICROSCOPYf_chiral_restr0.0481995
ELECTRON MICROSCOPYf_plane_restr0.0032295
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_11AELECTRON MICROSCOPYNCS constraints0
ens_12AELECTRON MICROSCOPYNCS constraints1.90183719429E-13
ens_13AELECTRON MICROSCOPYNCS constraints2.35988398406E-13
ens_14AELECTRON MICROSCOPYNCS constraints1.2207669448E-13

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