German Federal Ministry for Education and Research
03Z22HN22
Germany
German Federal Ministry for Education and Research
03Z22HN23
Germany
German Federal Ministry for Education and Research
03Z22HI2
Germany
European Regional Development Fund
ZS/2016/04/78115
European Union
European Regional Development Fund
European Union
Agence Nationale de la Recherche (ANR)
ME 4165/2-1
France
Agence Nationale de la Recherche (ANR)
KE 1478/7-1
France
Austrian Science Fund
I 5359-N
Austria
Citation
Journal: Biomacromolecules / Year: 2022 Title: Cryo-Electron Microscopy Snapshots of Eukaryotic Membrane Proteins in Native Lipid-Bilayer Nanodiscs. Authors: Kevin Janson / Fotis L Kyrilis / Christian Tüting / Marie Alfes / Manabendra Das / Toni K Träger / Carla Schmidt / Farzad Hamdi / Carolyn Vargas / Sandro Keller / Annette Meister / Panagiotis L Kastritis / Abstract: New technologies for purifying membrane-bound protein complexes in combination with cryo-electron microscopy (EM) have recently allowed the exploration of such complexes under near-native conditions. ...New technologies for purifying membrane-bound protein complexes in combination with cryo-electron microscopy (EM) have recently allowed the exploration of such complexes under near-native conditions. In particular, polymer-encapsulated nanodiscs enable the study of membrane proteins at high resolution while retaining protein-protein and protein-lipid interactions within a lipid bilayer. However, this powerful technology has not been exploited to address the important question of how endogenous─as opposed to overexpressed─membrane proteins are organized within a lipid environment. In this work, we demonstrate that biochemical enrichment protocols for native membrane-protein complexes from in combination with polymer-based lipid-bilayer nanodiscs provide a substantial improvement in the quality of recovered endogenous membrane-protein complexes. Mass spectrometry results revealed ∼1123 proteins, while multiple 2D class averages and two 3D reconstructions from cryo-EM data furnished prominent structural signatures. This integrated methodological approach to enriching endogenous membrane-protein complexes provides unprecedented opportunities for a deeper understanding of eukaryotic membrane proteomes.
Name: SB-DIBMA solubilized Chaetomium thermophilum membranes mMW fraction type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1 Details: Sample was created by solubilizing native chaetomium termophilum membranes with the aid of the copolymer SB-DIBMA
Details: Solutions were freshly prepared, sterile filtrated, and sonicated before usage
Grid
Model: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY
Vitrification
Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: Blotting time of 12 s Blotforce of 0.
Details
This sample was purified by size exclusion chromatography. Subsequently, multiple fractions in the medium molecular weight region were pooled together to obtain a higher protein concentration.
-
Electron microscopy
Microscope
TFS GLACIOS
Temperature
Min: 77.15 K / Max: 103.15 K
Alignment procedure
Coma free - Residual tilt: 14.7 mrad
Details
Grid screening was performed manually until criteria for good acquisition areas was narrowed down.
Image recording
Film or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4048 pixel / Digitization - Dimensions - Height: 4048 pixel / Number grids imaged: 1 / Number real images: 5912 / Average electron dose: 64.72 e/Å2
Electron beam
Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
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