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Locking down the SARS-CoV-2 spike Many efforts to develop therapies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are focused on the spike (S) protein trimer that binds to the host receptor. Structures of trimeric S protein show its receptor-binding domain in either an up or a down conformation. Toelzer et al. produced SARS-CoV-2 S in insect cells and determined the structure by cryo–electron microscopy. In their dataset, the closed form was predominant and was stabilized by binding linoleic acid, an essential fatty acid. A similar binding pocket appears to be present in previous highly pathogenic coronaviruses, and past studies suggested links between viral infection and fatty acid metabolism. The pocket could be exploited to develop inhibitors that trap S protein in the closed conformation. Science, this issue p. 725, The SARS-CoV-2 spike binds linoleic acid, a key molecule in inflammation, immune modulation, and membrane fluidity., Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), represents a global crisis. Key to SARS-CoV-2 therapeutic development is unraveling the mechanisms that drive high infectivity, broad tissue tropism, and severe pathology. Our 2.85-angstrom cryo–electron microscopy structure of SARS-CoV-2 spike (S) glycoprotein reveals that the receptor binding domains tightly bind the essential free fatty acid linoleic acid (LA) in three composite binding pockets. A similar pocket also appears to be present in the highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV). LA binding stabilizes a locked S conformation, resulting in reduced angiotensin-converting enzyme 2 (ACE2) interaction in vitro. In human cells, LA supplementation synergizes with the COVID-19 drug remdesivir, suppressing SARS-CoV-2 replication. Our structure directly links LA and S, setting the stage for intervention strategies that target LA binding by SARS-CoV-2.

Paul et al. use cryo-ET to show that microtubules can contain actin filaments in their lumen. Two types of F-actin structure are distinguished, Class I and Class II, with slightly different helical symmetries and outer microtubule diameters. They call these filaments microtubule lumenal actin (ML-actin)., Microtubules and filamentous (F-) actin engage in complex interactions to drive many cellular processes from subcellular organization to cell division and migration. This is thought to be largely controlled by proteins that interface between the two structurally distinct cytoskeletal components. Here, we use cryo-electron tomography to demonstrate that the microtubule lumen can be occupied by extended segments of F-actin in small molecule–induced, microtubule-based, cellular projections. We uncover an unexpected versatility in cytoskeletal form that may prompt a significant development of our current models of cellular architecture and offer a new experimental approach for the in situ study of microtubule structure and contents.

In order to sustain genomic stability and cellular viability, the ends of linear eukaryotic chromosomes are capped and protected by telomeres. The mechanism by which the length of telomeric DNA is maintained involves a specialized reverse transcriptase, called telomerase. This thesis focuses on the structural investigation of two proteins that play essential roles in the regulation of different stages of telomerase activity: TCAB1 and the CST complex. TCAB1 participates in the maturation process of the telomerase RNA subunit and is critical for telomerase trafficking in vivo. Mutations in TCAB1 lead to defects in telomere maintenance and give rise to severe forms of dyskeratosis congenita (DC). The CST complex, consisting of Ctc1, Stn1 and Ten1, limits telomerase activity in the late stages of telomere elongation. CST also initiates the fill-in synthesis of the complementary strand by recruiting the Polα/Primase complex. Mutations in CST are associated with Coat Plus, DC and related diseases. In this thesis, I present various strategies for the expression and purification of TCAB1 and the CST complex and structural analysis using negative stain electron microscopy (EM). Using primary sequence analysis and computational methods, I demonstrated that TCAB1 contains seven putative WD40 repeats within its central domain rather than the five published. Using various strategies for expression of TCAB1 in E.coli, that lead to aggregated protein, it was published that TCAB1 folding requires an elaborate machinery including the TCP-Ring Complex (TRiC) that is only present in higher eukaryotes. For the CST complex, I found that expression of full length Ctc1 in E.coli was not feasible and that it necessitated co-expression with Stn1 and Ten1. This was achieved by setting up MultiBac expression in insect cells. Results are presented for a purification strategy for obtaining a pure complex that contains stoichiometric amounts of the three proteins in mg quantities. As an initial stage to determining the cryo-EM structure, I present a low-resolution threedimensional structure (25Å) of the CST complex obtained using negative stain EM and single-particle reconstruction. Finally, I describe strategies to obtain a higher resolution structure bound to telomeric DNA. Doctor of Philosophy (SBS)