Substrate Effects on Structural Studies using Cryo-Em

Abstract

In electron microscopy, glow discharge has been widely used to render the carbon film hydrophilic for better adhesion of aqueous solution. However, the target macromolecules (e.g. proteins) may also interact with the charge on the carbon film, which may introduce preferred orientation and/or some other artifacts. A promising alternative is to use two-dimensional (2D) crystal as a substrate. This method has been successfully employed to study DNA molecules, proteoliposomes, and multiprotein complexes. Here we will use liposomes (i.e. lipid vesicles) as a model system to study the effect of substrate on targeted structures. Four types of substrates will be studied: untreated carbon film, glow-discharged carbon film, holey carbon film, and 2D streptavidin crystal. Liposomes, doped with a few copies of biotinylated lipid and osmotically swollen to ensure a spherical shape, will be allowed to attach to the substrate before blotting and rapid freezing of the specimen. Then cryo-electron microscopy of tilted specimens and cryo-electron tomography will be employed, and the resulted images and tomographs will be compared with spherical liposome models. The deformation of the liposomes will be used to quantify the effect of the substrate on the targeted structure.