In Situ Imaging and Structure Determination of Bacterial Toxin Delivery Systems Using Electron Cryotomography.

Determining the three-dimensional structure of biomacromolecules at high resolution in their native cellular environment is a major challenge for structural biology. Toward this end, electron cryotomography (ECT) allows large bio-macromolecular assemblies to be imaged directly in their hydrated physiological milieu to ~4 nm resolution. Combining ECT with other techniques like fluorescent imaging, immunogold labeling, and genetic manipulation has allowed the in situ investigation of complex biological processes at macromolecular resolution. Furthermore, the advent of cryogenic focused ion beam (FIB) milling has extended the domain of ECT to include regions even deep within thick eukaryotic cells. Anticipating two audiences (scientists who just want to understand the potential and general workflow involved and scientists who are learning how to do the work themselves), here we present both a broad overview of this kind of work and a step-by-step example protocol for ECT and subtomogram averaging using the Legionella pneumophila Dot/Icm type IV secretion system (T4SS) as a case study. While the general workflow is presented in step-by-step detail, we refer to online tutorials, user's manuals, and other training materials for the essential background understanding needed to perform each step.