Cellular architecture of Treponema pallidum: novel flagellum, periplasmic cone, and cell envelope as revealed by cryo electron tomography.

High-resolution cryo electron tomography (cryo-ET) was utilized to visualize Treponema pallidum, the causative agent of syphilis, at the molecular level. Three-dimensional (3D) reconstructions from 304 infectious organisms revealed unprecedented cellular structures of this unusual member of the spirochetal family. High-resolution cryo-ET reconstructions provided detailed structures of the cell envelope, which is significantly different from that of Gram-negative bacteria. The 4-nm lipid bilayer of both outer membrane and cytoplasmic membrane resolved in 3D reconstructions, providing an important marker for interpreting membrane-associated structures. Abundant lipoproteins cover the outer leaflet of the cytoplasmic membrane, in contrast to the rare outer membrane proteins visible by scanning probe microscopy. High-resolution cryo-ET images also provided the first observation of T. pallidum chemoreceptor arrays, as well as structural details of the periplasmically located cone-shaped structure at both ends of the bacterium. Furthermore, 3D subvolume averages of periplasmic flagellar motors and flagellar filaments from living organisms revealed the novel flagellar architectures that may facilitate their rotation within the confining periplasmic space. Our findings provide the most detailed structural understanding of periplasmic flagella and the surrounding cell envelope, which enable this enigmatic bacterium to efficiently penetrate tissue and to escape host immune responses.

Structure of the yeast vacuolar ATPase.

The subunit architecture of the yeast vacuolar ATPase (V-ATPase) was analyzed by single particle transmission electron microscopy and electrospray ionization (ESI) tandem mass spectrometry. A three-dimensional model of the intact V-ATPase was calculated from two-dimensional projections of the complex at a resolution of 25 angstroms. Images of yeast V-ATPase decorated with monoclonal antibodies against subunits A, E, and G position subunit A within the pseudo-hexagonal arrangement in the V1, the N terminus of subunit G in the V1-V0 interface, and the C terminus of subunit E at the top of the V1 domain. ESI tandem mass spectrometry of yeast V1-ATPase showed that subunits E and G are most easily lost in collision-induced dissociation, consistent with a peripheral location of the subunits. An atomic model of the yeast V-ATPase was generated by fitting of the available x-ray crystal structures into the electron microscopy-derived electron density map. The resulting atomic model of the yeast vacuolar ATPase serves as a framework to help understand the role the peripheral stalk subunits are playing in the regulation of the ATP hydrolysis driven proton pumping activity of the vacuolar ATPase.

A structural model of the vacuolar ATPase from transmission electron microscopy.

Vacuolar ATPases (V-ATPases) are large, membrane bound, multisubunit protein complexes which function as ATP hydrolysis driven proton pumps. V-ATPases and related enzymes are found in the endomembrane system of eukaryotic organsims, the plasma membrane of specialized cells in higher eukaryotes, and the plasma membrane of prokaryotes. The proton pumping action of the vacuolar ATPase is involved in a variety of vital intra- and inter-cellular processes such as receptor mediated endocytosis, protein trafficking, active transport of metabolites, homeostasis and neurotransmitter release. This review summarizes recent progress in the structure determination of the vacuolar ATPase focusing on studies by transmission electron microscopy. A model of the subunit architecture of the vacuolar ATPase is presented which is based on the electron microscopic images and the available information from genetic, biochemical and biophysical experiments.