German Federal Ministry for Education and Research
03Z22HN22
ドイツ
German Federal Ministry for Education and Research
03Z22HN23
ドイツ
German Federal Ministry for Education and Research
03Z22HI2
ドイツ
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
フランス
Agence Nationale de la Recherche (ANR)
KE 1478/7-1
フランス
Austrian Science Fund
I 5359-N
オーストリア
引用
ジャーナル: Biomacromolecules / 年: 2022 タイトル: Cryo-Electron Microscopy Snapshots of Eukaryotic Membrane Proteins in Native Lipid-Bilayer Nanodiscs. 著者: 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 / 要旨: 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.
名称: SB-DIBMA solubilized Chaetomium thermophilum membranes mMW fraction タイプ: complex / ID: 1 / キメラ: Yes / 親要素: 0 / 含まれる分子: #1 詳細: Sample was created by solubilizing native chaetomium termophilum membranes with the aid of the copolymer SB-DIBMA
凍結剤: ETHANE / チャンバー内湿度: 95 % / チャンバー内温度: 277.15 K / 装置: FEI VITROBOT MARK IV / 詳細: Blotting time of 12 s Blotforce of 0.
詳細
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.
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電子顕微鏡法
顕微鏡
TFS GLACIOS
温度
最低: 77.15 K / 最高: 103.15 K
アライメント法
Coma free - Residual tilt: 14.7 mrad
詳細
Grid screening was performed manually until criteria for good acquisition areas was narrowed down.