Rapid tilt-series method for cryo-electron tomography: Characterizing stage behavior during FISE acquisition

We and others recently developed rapid tilt-series acquisition methods for cryo-electron tomography on a Titan Krios G3i equipped with a single axis holder and a K-series direct electron detector and showed that one of these, the fast-incremental single exposure (FISE) method, significantly accelerates tilt-series acquisition when compared to traditional methods while preserving the quality of the images. Here, we characterize the behavior of our single axis holder in detail during a FISE experiment to optimally balance data quality with speed. We explain our methodology in detail so others can characterize their own stages, and conclude with recommendations for projects with different resolution goals

Electron cryotomography: a new view into microbial ultrastructure

Electron cryotomography (ECT) is an emerging technology that allows thin samples such as small bacterial cells to be imaged in 3D in a nearly native state to ‘molecular’ (not, vert, similar4 nm) resolution. As such, ECT is beginning to deliver long-awaited insight into the positions and structures of cytoskeletal filaments, cell wall elements, motility machines, chemoreceptor arrays, internal compartments, and other ultrastructures. Here we briefly explain ECT, review its recent contributions to microbiology, and conclude with a discussion of future prospects

The bacterial cytoskeleton: more than twisted filaments

Far from being simple ‘bags’ of enzymes, bacteria are richly endowed with ultrastructures that challenge and expand standard definitions of the cytoskeleton. Here we review rods, rings, twisted pairs, tubes, sheets, spirals, moving patches, meshes and composites, and suggest defining the term ‘bacterial cytoskeleton’ as all cytoplasmic protein filaments and their superstructures that move or scaffold (stabilize/position/recruit) other cellular materials. The evolution of each superstructure has been driven by specific functional requirements. As a result, while homologous proteins with different functions have evolved to form surprisingly divergent superstructures, those of unrelated proteins with similar functions have converged

A new view into prokaryotic cell biology from electron cryotomography

Electron cryotomography (ECT) enables intact cells to be visualized in 3D in an essentially native state to 'macromolecular' (~4 nm) resolution, revealing the basic architectures of complete nanomachines and their arrangements in situ. Since its inception, ECT has advanced our understanding of many aspects of prokaryotic cell biology, from morphogenesis to subcellular compartmentalization and from metabolism to complex interspecies interactions. In this Review, we highlight how ECT has provided structural and mechanistic insights into the physiology of bacteria and archaea and discuss prospects for the future

Rapid Tilt-Series Method for Cryo-Electron Tomography: Characterizing Stage Behavior During FISE Acquisition

We and others recently developed rapid tilt-series acquisition methods for cryo-electron tomography on a Titan Krios G3i equipped with a single axis holder and a K-series direct electron detector and showed that one of these, the fast-incremental single exposure (FISE) method, significantly accelerates tilt-series acquisition when compared to traditional methods while preserving the quality of the images. Here, we characterize the behavior of our single axis holder in detail during a FISE experiment to optimally balance data quality with speed. We explain our methodology in detail so others can characterize their own stages, and conclude with recommendations for projects with different resolution goals