Streptavidin Affinity Grids for cryo-EM

Abstract

The preparation of specimen grids for cryo-EM often proves to be difficult because of unwanted factors such as preferential orientation of particles, too few particles being seen within holes, particle disruption occurring within thin aqueous films, and unexpected aggregation of sample material. In instances where it is suspected that those difficulties might be caused by interaction with the air-water interface, a possible solution is to immobilize particles on a structure-friendly support film, thus preventing diffusion to the air-water interface of the thin aqueous film that remains after blotting away excess sample. We have developed a form of EM grid on which streptavidin-monolayer crystals serve as an affinity support-film. A thin covering of trehalose is used to embed the streptavidin monolayers during storage, thereby preserving the crystalline structure and biotin-binding function of the streptavidin. These affinity grids can thus be prepared in any desired quantity and stored for future use. Many strategies are possible for immobilizing particles by affinity-binding to the streptavidin monolayer after washing off the protective layer of trehalose. One of the simplest is to randomly biotinylate one or two surface-lysine residues on particles of interest. Advantages of these grids include: (1) a good surface density of immobilized particles is obtained rapidly at sample concentrations as low as 50 nM; (2) the streptavidin component of the image serves as an internal standard to verify sample preservation and image quality; and (3) the streptavidin “background” in the image can be removed, whenever desired, by Fourier filtering.