Cryo EM Study of Protein Complexes Bound to Streptavidin Crystal Layers

Abstract

Conventional cryo-EM specimens use either continuous or holey, glow-discharge treated carbon films to prepare samples. As a result, particles are either immobilized by binding to an unnatural (i.e. carbon-film) surface, or they collide multiple times (due to rapid diffusion) with the freshly blotted air-water interface. In either case there is a risk that considerable structural heterogeneity may occur, or even that particles may undergo severe denaturation.Binding biotinylated particles to streptavidin monolayer crystals is a more structure-friendly way to immobilize particles. We have developed a protocol for preparing biotinylated protein complexes and tethering them to the surface of streptavidin monolayer-crystals. Three different multi-protein complexes have been used to demonstrate the generality of this method. The biotinylated protein complexes bind to the streptavidin crystal layer with high affinity, whereas the unbiotinylated control samples do not do so. In addition, the same technique can be used to produce a uniform distribution of Euler angles. The thermosomes, known to adopt a preferred orientation with other methods of cryo-EM specimen preparation, have been used as a test sample. Indeed, the results confirm that a uniform distribution of Euler angles is obtained for the biotinylated thermosome. We thus conclude that biotinylation, together with binding to 2-D crystals of streptavidin, is a better method to prepare protein complexes for cryo-EM study.