Electron cryotomography (ECT) can reveal the native structure and arrangement of macromolecular complexes inside intact cells. This technique has greatly advanced our understanding of the ultrastructure of bacterial cells. We now view bacteria as structurally complex assemblies of macromolecular machines rather than as undifferentiated bags of enzymes. To date, our group has applied ECT to nearly 90 different bacterial species, collecting more than 15,000 cryotomograms. In addition to known structures, we have observed, to our knowledge, several uncharacterized features in these tomograms. Some are completely novel structures; others expand the features or species range of known structure types. Here, we present a survey of these uncharacterized bacterial structures in the hopes of accelerating their identification and study, and furthering our understanding of the structural complexity of bacterial cells.IMPORTANCE Bacteria are more structurally complex than is commonly appreciated. Here we present a survey of previously uncharacterized structures that we observed in bacterial cells by electron cryotomography, structures that will initiate new lines of research investigating their identities and roles.KEYWORDS bacteria, electron cryotomography, bacterial ultrastructure, uncharacterized structures, electron microscopy T he history of cell biology has been punctuated by advances in imaging technology. In particular, the development of electron microscopy (EM) in the 1930s produced a wealth of new information about the ultrastructure of cells (1). For the first time, the structure of cell envelopes, internal organelles, cytoskeletal filaments, and even large macromolecular complexes like ribosomes became visible. A further advance came in the 1980s and 1990s with the development of electron cryotomography (ECT) (2), which allows small cells to be imaged intact in 3D in a near-native, "frozen-hydrated" state to "macromolecular" (ϳ5 nm) resolution, without the limitations and artifacts of more traditional specimen-preparation methods (3).ECT has helped reveal the previously unappreciated complexity of "simple" bacterial cells. Our group has been using ECT to study bacteria for more than a decade, generating more than 15,000 tomograms of 88 different species. These tomograms have revealed new insights into, among other things, the bacterial cytoskeleton (4-7), cell wall architecture (8, 9), morphogenesis (10), metabolism (11), motility (12-15),

23 24 Running title: ECT of novel bacterial structures 25 26 Keywords: bacteria, electron cryotomography, cryo-EM, bacterial ultrastructure, 27 uncharacterized structures 28 29 30 2 SUMMARY STATEMENT 31 Here we present a survey of previously uncharacterized structures we have observed in bacterial 32 cells by electron cryotomography, in the hopes of spurring their identification and study. 33 34 ABSTRACT 35 Electron cryotomography (ECT) can reveal the native structure and arrangement of 36 macromolecular complexes inside intact cells. This technique has greatly advanced our 37understanding of the ultrastructure of bacterial cells. Rather than undifferentiated bags of 38 enzymes, we now view bacteria as structurally complex assemblies of macromolecular 39 machines. To date, our group has applied ECT to nearly 90 different bacterial species, collecting 40 more than 15,000 cryotomograms. In addition to known structures, we have observed several, to 41 our knowledge, uncharacterized features in these tomograms. Some are completely novel 42 structures; others expand the features or species range of known structure types. Here we present 43 a survey of these uncharacterized bacterial structures in the hopes of accelerating their 44 identification and study, and furthering our understanding of the structural complexity of 45 bacterial cells. 46