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- EMDB-23207: Unliganded ELIC in styrene-maleic-acid nanodiscs at 2.5-Angstrom ... -

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Basic information

Entry
Database: EMDB / ID: EMD-23207
TitleUnliganded ELIC in styrene-maleic-acid nanodiscs at 2.5-Angstrom resolution
Map data
SampleUnliganded Erwinia chrysanthemi ligand gated ion channel in styrene-maleic-acid nanodiscs
  • Gamma-aminobutyric-acid receptor subunit beta-1
  • (ligand) x 2
Function / homology
Function and homology information


extracellular ligand-gated ion channel activity / transmembrane signaling receptor activity / integral component of membrane / identical protein binding
Similarity search - Function
Neurotransmitter-gated ion-channel transmembrane domain superfamily / 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) / Dickeya dadantii (strain 3937) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsGrosman C / Kumar P
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-
UpdateJul 14, 2021-
Current statusJul 14, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.7
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.7
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7l6q
  • Surface level: 0.7
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_23207.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 256 pix.
= 274.688 Å
1.07 Å/pix.
x 256 pix.
= 274.688 Å
1.07 Å/pix.
x 256 pix.
= 274.688 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.073 Å
Density
Contour LevelBy AUTHOR: 0.7 / Movie #1: 0.7
Minimum - Maximum0.0 - 11.148251
Average (Standard dev.)0.012643953 (±0.22475502)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 274.688 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0731.0731.073
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z274.688274.688274.688
α/β/γ90.00090.00090.000
start NX/NY/NZ192139186
NX/NY/NZ211274246
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean0.00011.1480.013

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Supplemental data

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Segmentation: #1

Fileemd_23207_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Half-map 1

Fileemd_23207_half_map_1.map
AnnotationHalf-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Half-map 2

Fileemd_23207_half_map_2.map
AnnotationHalf-map 2
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

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Sample components

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Entire Unliganded Erwinia chrysanthemi ligand gated ion channel in styre...

EntireName: Unliganded Erwinia chrysanthemi ligand gated ion channel in styrene-maleic-acid nanodiscs
Number of Components: 4

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Component #1: protein, Unliganded Erwinia chrysanthemi ligand gated ion channel...

ProteinName: Unliganded Erwinia chrysanthemi ligand gated ion channel in styrene-maleic-acid nanodiscs
Recombinant expression: No
MassTheoretical: 184.2 MDa
SourceSpecies: Dickeya dadantii (bacteria)
Source (engineered)Expression System: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Strain: BL21

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Component #2: protein, Gamma-aminobutyric-acid receptor subunit beta-1

ProteinName: Gamma-aminobutyric-acid receptor subunit beta-1 / Number of Copies: 5 / Recombinant expression: No
MassTheoretical: 36.879 kDa
SourceSpecies: Dickeya dadantii (strain 3937) (bacteria) / Strain: 3937
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #3: ligand, (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phos...

LigandName: (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]oxy}-1-[(hexadecanoyloxy)methyl]ethyl (9Z)-octadec-9-enoate
Number of Copies: 5 / Recombinant expression: No
MassTheoretical: 0.749007 kDa

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Component #4: ligand, CARDIOLIPIN

LigandName: CARDIOLIPIN / Number of Copies: 5 / Recombinant expression: No
MassTheoretical: 1.464043 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen State: Particle / Method: cryo EM
Sample solutionpH: 8
VitrificationInstrument: LEICA EM GP / Cryogen Name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron Source: FIELD EMISSION GUN / Accelerating Voltage: 300 kV / Electron Dose: 56.23 e/Å2 / Illumination Mode: SPOT SCAN
LensImaging Mode: BRIGHT FIELD
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied Symmetry: C5 (5回回転対称) / Number of Projections: 1239532
3D reconstructionResolution: 2.5 Å / Resolution Method: FSC 0.143 CUT-OFF

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Atomic model buiding

Output model

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