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

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. 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.

Lipid exchange among electroneutral Sulfo-DIBMA nanodiscs is independent of ion concentration

“…The average hydrodynamic size of labeled nanodiscs as determined by DLS and the associated standard deviation amounted to (8.90 ± 1.47) nm ( n = 5), again in good agreement with previous values determined for Sulfo-DIBMA nanodiscs by DLS and other methods ( Glueck et al. 2022 ; Janson et al. 2022 ).…”

“…2022 ), negative-stain electron microscopy (nsEM) ( Glueck et al. 2022 ; Janson et al. 2022 ), and cryoEM ( Janson et al.…”

“…2022 ). Finally, Sulfo-DIBMA nanodiscs have successfully been used in cryogenic electron microscopy (cryoEM) studies of endogenous eukaryotic membrane proteins, thereby outperforming other nanodisc-forming polymers in terms of protein-extraction efficiency and sample quality ( Janson et al. 2022 ).…”

Effect of Cholesterol on the Structure and Composition of Glyco-DIBMA Lipid Particles

“…Glyco-DIBMA retains solubility similar to that of DIBMA, a similar UV absorption spectrum, and a smaller amount of negative charge. Indeed, the electrostatic repulsion between the polymer chains has been suggested to induce the broad size distribution and larger size of DIBMALPs compared to those of SMALPs, Glyco-DIBMALPs, and other neutral polymers. ,− As recently reported, Glyco-DIBMALPs are stable at physiological pH and ionic strength and in the presence of divalent cations. The formation of Glyco-DIBMALPs with different synthetic phospholipids was tested together with the application of this co-polymer for the direct extraction of membrane proteins from Escherichia coli cells …”

Cancer-Associated Gangliosides as a Therapeutic Target for Host Defense Peptide Mimics

“…42 When the fractional area change is 0.04, the rate constant of pore formation is extremely high, though this value inducing significant pore formation depends on the types of lipids. Future experiments using methods like NMR, 43 crystallography, 44 circular dichroism, 45 electron microscopy, 46 surface plasmon resonance, 47 and molecular dynamics simulations 48 might offer a clearer understanding of OAK's specific mechanism against cancer cells.…”