Location and architecture of the Caulobacter crescentus chemoreceptor array.

A new method for recording both fluorescence and cryo-EM images of small bacterial cells was developed and used to identify chemoreceptor arrays in cryotomograms of intact Caulobacter crescentus cells. We show that in wild-type cells preserved in a near-native state, the chemoreceptors are hexagonally packed with a lattice spacing of 12 nm, just a few tens of nanometers away from the flagellar motor that they control. The arrays were always found on the convex side of the cell, further demonstrating that Caulobacter cells maintain dorsal/ventral as well as anterior/posterior asymmetry. Placing the known crystal structure of a trimer of receptor dimers at each vertex of the lattice accounts well for the density and agrees with other constraints. Based on this model for the arrangement of receptors, there are between one and two thousand receptors per array.

The structure of isolated Synechococcus strain WH8102 carboxysomes as revealed by electron cryotomography.

Carboxysomes are organelle-like polyhedral bodies found in cyanobacteria and many chemoautotrophic bacteria that are thought to facilitate carbon fixation. Carboxysomes are bounded by a proteinaceous outer shell and filled with ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the first enzyme in the CO(2) fixation pathway, but exactly how they enhance carbon fixation is unclear. Here we report the three-dimensional structure of purified carboxysomes from Synechococcus species strain WH8102 as revealed by electron cryotomography. We found that while the sizes of individual carboxysomes in this organism varied from 114 nm to 137 nm, surprisingly, all were approximately icosahedral. There were on average approximately 250 RuBisCOs per carboxysome, organized into three to four concentric layers. Some models of carboxysome function depend on specific contacts between individual RuBisCOs and the shell, but no evidence of such contacts was found: no systematic patterns of connecting densities or RuBisCO positions against the shell's presumed hexagonal lattice could be discerned, and simulations showed that packing forces alone could account for the layered organization of RuBisCOs.

Multiple large filament bundles observed in Caulobacter crescentus by electron cryotomography.

While the absence of any cytoskeleton was once recognized as a distinguishing feature of prokaryotes, it is now clear that a number of different bacterial proteins do form filaments in vivo. Despite the critical roles these proteins play in cell shape, genome segregation and cell division, molecular mechanisms have remained obscure in part for lack of electron microscopy-resolution images where these filaments can be seen acting within their cellular context. Here, electron cryotomography was used to image the widely studied model prokaryote Caulobacter crescentus in an intact, near-native state, producing three-dimensional reconstructions of these cells with unprecedented clarity and fidelity. We observed many instances of large filament bundles in various locations throughout the cell and at different stages of the cell cycle. The bundles appear to fall into four major classes based on shape and location, referred to here as 'inner curvature', 'cytoplasmic', 'polar' and 'ring-like'. In an attempt to identify at least some of the filaments, we imaged cells where crescentin and MreB filaments would not be present. The inner curvature and cytoplasmic bundles persisted, which together with their localization patterns, suggest that they are composed of as-yet unidentified cytoskeletal proteins. Thus bacterial filaments are frequently found as bundles, and their variety and abundance is greater than previously suspected.

Electron cryotomography sample preparation using the Vitrobot.

Electron cryotomography is the highest-resolution structural technique currently available that can be applied to unique objects such as flexible large protein complexes, irregular viruses, organelles and small cells. Specimens are preserved in a near-native, 'frozen-hydrated' state by vitrification. The thickness of the vitreous ice must be optimized for each specimen, and gold fiducials are typically added to facilitate image alignment. Here, we describe in detail our protocols for electron cryotomography sample preparation including (i) introduction of fiducial markers into the sample and (ii) sample vitrification. Because we almost exclusively use an automated, climate-controlled plunge-freezing device (the FEI Vitrobot) to vitrify our samples, we discuss its operation and parameters in detail. A session in which eight grids are prepared takes 1.5-2 h.

A "flip-flop" rotation stage for routine dual-axis electron cryotomography.

Electron cryotomography can be used to solve the three-dimensional structures of individual large macromolecules, assemblies, and even small intact cells to medium (approximately 4-8 nm) resolution in a near-native state, but restrictions in the range of accessible views are a major limitation. Here we report on the design, characterization, and demonstration of a new "flip-flop" rotation stage that allows facile and routine collection of two orthogonal tilt-series of cryosamples. Single- and dual-axis tomograms of a variety of samples are compared to illustrate qualitatively the improvement produced by inclusion of the second tilt-series. Exact quantitative expressions are derived for the volume of the remaining "missing pyramid" in reciprocal space. When orthogonal tilt-series are recorded to +/-65 degrees in each direction, as this new cryostage permits, only 11% of reciprocal space is left unmeasured. The tomograms suggest that further improvement could be realized, however, through better software to align and merge dual-axis tilt-series of cryosamples.