The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is required for cell entry and is the major focus for vaccine development. Here, we combine cryo electron tomography, subtomogram averaging and molecular dynamics simulations to structurally analyze S in situ. Compared to recombinant S, the viral S was more heavily glycosylated and occurred mostly in the closed pre-fusion conformation. We show that the stalk domain of S contains three hinges, giving the head unexpected orientational freedom. We propose that the hinges allow S to scan the host cell surface, shielded from antibodies by an extensive glycan coat. The structure of native S contributes to our understanding of SARS-CoV-2 infection and the development of safe vaccines.

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is required for cell entry and is the major focus for vaccine development. We combine cryo electron tomography, subtomogram averaging and molecular dynamics simulations to structurally analyze S in situ. Compared to recombinant S, the viral S is more heavily glycosylated and occurs predominantly in a closed pre-fusion conformation. We show that the stalk domain of S contains three hinges that give the globular domain unexpected orientational freedom. We propose that the hinges allow S to scan the host cell surface, shielded from antibodies by an extensive glycan coat. The structure of native S contributes to our understanding of SARS-CoV-2 infection and the development of safe vaccines. The large scale tomography data set of SARS-CoV-2 used for this study is therefore sufficient to resolve structural features to below 5 Ångstrom, and is publicly available at EMPIAR-10453.

SummaryThe molecular events that direct nuclear pore complex (NPC) assembly toward nuclear envelopes have been conceptualized in two pathways that occur during mitosis or interphase, respectively. In gametes and embryonic cells, NPCs also occur within stacked cytoplasmic membrane sheets, termed annulate lamellae (AL), which serve as NPC storage for early development. The mechanism of NPC biogenesis at cytoplasmic membranes remains unknown. Here, we show that during Drosophila oogenesis, Nucleoporins condense into different precursor granules that interact and progress into NPCs. Nup358 is a key player that condenses into NPC assembly platforms while its mRNA localizes to their surface in a translation-dependent manner. In concert, Microtubule-dependent transport, the small GTPase Ran and nuclear transport receptors regulate NPC biogenesis in oocytes. We delineate a non-canonical NPC assembly mechanism that relies on Nucleoporin condensates and occurs away from the nucleus under conditions of cell cycle arrest.

The faithful assembly of protein complexes in space and time is a hallmark of cellular homeostasis. Complex assembly might be seeded already during translation, if interacting subunits are recruited to the nascent chain. Here, we review recent discoveries suggesting that such cotranslational assembly is a prominent feature throughout the proteome. It might contribute to the efficiency and efficacy of assembly and occurs in coordination rather than competition with chaperones. We discuss how cotranslational assembly structurally contributes to the organizational order of assembly pathways and their surveillance. Taken together, these novel insights suggest that cotranslational assembly is intimately linked with the regulation of protein abundance, stability, and activity, offering an attractive explanation for many cellular phenomena. Coordinating Protein Complex AssemblyProtein complexes are a key organizational unit of the proteome. The assembly of such complexes is a nontrivial task in the crowded interior of a cell, where each protein is in frequent contact with other macromolecules and therefore in competition for binding partners. Inevitably, cells had to come up with strategies to ensure faithful and efficient assembly. For many complexes, assembly based on the random collision of subunits is sufficient, as evidenced by assembly in vitro [1,2]. Others rely on the standard suite of cellular broad-specificity chaperones [3], have evolved dedicated chaperones [4], or even entire assembly organelles, as exemplified by ribosomal assembly in the nucleolus [5]. In all cases, premature or unintended interactions of nascent peptides are prevented, either by cotranslational binding of chaperones or by active transport to a suitable environment. There is, however, another way to achieve this; namely, through immediate cotranslational folding and concomitant association of binding partners.