cryoWriter: a blotting free cryo-EM preparation system with a climate jet and cover-slip injector

Electron microscopy (EM) introduced a fast and lasting change to structural and cellular biology. However, the sample preparation is still the bottleneck in the cryogenic electron microscopy (cryo-EM) workflow. Classical specimen preparation methods employ a harsh paper-blotting step, and the protein particles are exposed to a damaging air-water interface. Therefore, improved preparation strategies are urgently needed. Here, we present an amended microfluidic sample preparation method, which entirely avoids paper blotting and allows the passivation of the air-water interface during the preparation process. First, a climate jet excludes oxygen from the sample environment and controls the preparation temperature by varying the relative humidity of the grid environment. Second, the integrated "coverslip injector" allows the modulation of the air-water interface of the thin sample layer with effector molecules. We will briefly discuss the climate jet's effect on the stability and dynamics of the sample thin films. Furthermore, we will address the coverslip injector and demonstrate significant improvement in the sample quality

Electron microscopy for ultrastructural analysis and protein localization in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is a key model system for studying of a multitude of cellular processes because of its amenability to genetics, molecular biology and biochemical procedures. Ultrastructural examinations of this organism, though, are traditionally difficult because of the presence of a thick cell wall and the high density of cytoplasmic proteins. A series of recent methodological and technical developments, however, has revived interest in morphological analyses of yeast (e.g. 123). Here we present a review of established and new methods, from sample preparation to imaging, for the ultrastructural analysis of S. cerevisiae. We include information for the use of different fixation methods, embedding procedures, approaches for contrast enhancement, and sample visualization techniques, with references to successful examples. The goal of this review is to guide researchers that want to investigate a particular process at the ultrastructural level in yeast by aiding in the selection of the most appropriate approach to visualize a specific structure or subcellular compartment

Structural investigation of ACE2 dependent disassembly of the trimeric SARS-CoV-2 Spike glycoprotein

The human membrane protein Angiotensin-converting enzyme 2 (hACE2) acts as the main receptor for host cells invasion of the new coronavirus SARS-CoV-2. The viral surface glycoprotein Spike binds to hACE2, which triggers virus entry into cells. As of today, the role of hACE2 for virus fusion is not well understood. Blocking the transition of Spike from its prefusion to post-fusion state might be a strategy to prevent or treat COVID-19. Here we report a single particle cryo-electron microscopy analysis of SARS-CoV-2 trimeric Spike in presence of the human ACE2 ectodomain. The binding of purified hACE2 ectodomain to Spike induces the disassembly of the trimeric form of Spike and a structural rearrangement of its S1 domain to form a stable, monomeric complex with hACE2. This observed hACE2 dependent dissociation of the Spike trimer suggests a mechanism for the therapeutic role of recombinant soluble hACE2 for treatment of COVID-19