Cryo-Electron Microscopy Snapshots of Eukaryotic Membrane Proteins in Native Lipid-Bilayer Nanodiscs

• Published in: Biomacromolecules 2022-12, Vol.23 (12), p.5084-5094
• Main Authors: Janson, Kevin, Kyrilis, Fotis L., Tüting, Christian, Alfes, Marie, Das, Manabendra, Träger, Toni K., Schmidt, Carla, Hamdi, Farzad, Vargas, Carolyn, Keller, Sandro, Meister, Annette, Kastritis, Panagiotis L.
• Format: Article
• Language: English
• Subjects: Cryoelectron Microscopy - methods; Eukaryota - metabolism; Lipid Bilayers - chemistry; Membrane Proteins - chemistry; Nanostructures - chemistry; Polymers - chemistry
• ISSN: 1525-7797; 1526-4602
• DOI: 10.1021/acs.biomac.2c00935

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 Chaetomium thermophilum 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.