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

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

Growth and Localization of Polyhydroxybutyrate Granules in Ralstonia eutropha

The bacterium Ralstonia eutropha forms cytoplasmic granules of polyhydroxybutyrate that are a source of biodegradable thermoplastic. While much is known about the biochemistry of polyhydroxybutyrate production, the cell biology of granule formation and growth remains unclear. Previous studies have suggested that granules form either in the inner membrane, on a central scaffold, or in the cytoplasm. Here we used electron cryotomography to monitor granule genesis and development in 3 dimensions (3-D) in a near-native, “frozen-hydrated” state in intact Ralstonia eutropha cells. Neither nascent granules within the cell membrane nor scaffolds were seen. Instead, granules of all sizes resided toward the center of the cytoplasm along the length of the cell and exhibited a discontinuous surface layer more consistent with a partial protein coating than either a lipid mono- or bilayer. Putatively fusing granules were also seen, suggesting that small granules are continually generated and then grow and merge. Together, these observations support a model of biogenesis wherein granules form in the cytoplasm coated not by phospholipid but by protein. Previous thin-section electron microscopy (EM), fluorescence microscopy, and atomic force microscopy (AFM) results to the contrary may reflect both differences in nucleoid condensation and specimen preparation-induced artifacts

Fusion of DARPin to Aldolase Enables Visualization of Small Protein by Cryo-EM

Solving protein structures by single-particle cryoelectron microscopy (cryo-EM) has become a crucial tool in structural biology. While exciting progress is being made toward the visualization of small macromolecules, the median protein size in both eukaryotes and bacteria is still beyond the reach of cryo-EM. To overcome this problem, we implemented a platform strategy in which a small protein target was rigidly attached to a large, symmetric base via a selectable adapter. Of our seven designs, the best construct used a designed ankyrin repeat protein (DARPin) rigidly fused to tetrameric rabbit muscle aldolase through a helical linker. The DARPin retained its ability to bind its target: GFP. We solved the structure of this complex to 3.0 Å resolution overall, with 5–8 Å resolution in the GFP region. As flexibility in the DARPin position limited the overall resolution of the target, we describe strategies to rigidify this element