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Bacterial secretion systems all tackle the challenge of secreting substrates from the cytoplasm out of the cell. Many unrelated secretion systems have evolved, strikingly diverse in their structures and mechanisms, from the small pump-like type II secretion systems to the colossal harpoon-like type VI secretion systems. What makes secretion systems intriguing also makes them difficult to study, however: they are cell envelope-associated, frequently contain moving or transiently associated components, and are very large.Cryogenic electron microscopy (CryoEM) is ideally poised to study secretion systems. CryoEM excels at determining the structures of large protein complexes, can discern different conformational states, and can image structures in situ. CryoEM has come of age in the past half-decade following a spectacular resolution revolution that changed cryoEM from low-resolution "blobology" to a methodology that can compete with X-ray crystallography and NMR for structure determination of biological molecules (Kühlbrandt, 2014). Here, we provide a primer for microbiologists on these capabilities. We describe its basic principles, its two main forms, and what the two forms can and cannot achieve. We then highlight the contributions of cryoEM to understanding how the diverse microbial secretion systems work, focusing on type III secretion systems. We end with a forward-facing overview of future prospects. | Cr yoEMCryoEM is a family of techniques that determine the three-dimensional (3-D) structures of biological molecules from two-dimensional (2-D) electron micrographs. In basic terms, this is achieved by imaging the specimen from many different angles to facilitate piecing together

The CST complex is a key player in telomere replication and stability, which in yeast comprises Cdc13, Stn1 and Ten1. While Stn1 and Ten1 are very well conserved across species, Cdc13 does not resemble its mammalian counterpart CTC1 either in sequence or domain organization, and Cdc13 but not CTC1 displays functions independently of the rest of CST. Whereas the structures of human CTC1 and CST have been determined, the molecular organization of Cdc13 remains poorly understood. Here, we dissect the molecular architecture of Candida glabrata Cdc13 and show how it regulates binding to telomeric sequences. Cdc13 forms dimers through the interaction between OB-fold 2 (OB2) domains. Dimerization stimulates binding of OB3 to telomeric sequences, resulting in the unfolding of ssDNA secondary structure. Once bound to DNA, Cdc13 prevents the refolding of ssDNA by mechanisms involving all domains. OB1 also oligomerizes, inducing higher-order complexes of Cdc13 in vitro. OB1 truncation disrupts these complexes, affects ssDNA unfolding and reduces telomere length in C. glabrata. Together, our results reveal the molecular organization of C. glabrata Cdc13 and how this regulates the binding and the structure of DNA, and suggest that yeast species evolved distinct architectures of Cdc13 that share some common principles.

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