Abstract
Liquid-electron microscopy (EM), the room-temperature correlate to cryo-EM, is a rapidly growing field providing high-resolution insights of macromolecules in solution. Here, we describe how liquid-EM experiments can incorporate automated tools to propel the field to new heights. We demonstrate fresh workflows for specimen preparation, data collection, and computing processes to assess biological structures in liquid. Adeno-associated virus (AAV) and the SARS-CoV-2 nucleocapsid (N) were used as model systems to highlight the technical advances. These complexes were selected based on their major differences in size and natural symmetry. AAV is a highly symmetric, icosahedral assembly with a particle diameter of ~25 nm. At the other end of the spectrum, N protein is an asymmetric monomer or dimer with dimensions of approximately 5–7 nm, depending upon its oligomerization state. Equally important, both AAV and N protein are popular subjects in biomedical research due to their high value in vaccine development and therapeutic efforts against COVID-19. Overall, we demonstrate how automated practices in liquid-EM can be used to decode molecules of interest for human health and disease.
Highlights
Understanding life’s processes in exquisite detail has been a work-in-progress over the last century
If smaller macromolecules are being evaluated for single-particle structural studies, similar to cryo-electron microscopy (EM) practices, thinner liquid layers offer a better signal-to-noise ratio (SNR) in TEM images
Recent work showed that virus particles in liquid were remarkably stable under lowdose conditions, producing structures with spatial resolutions on par with cryo-EM (Jonaid et al, 2021)
Summary
Understanding life’s processes in exquisite detail has been a work-in-progress over the last century. Due to recent advances in molecular microscopy, in particular cryo-electron microscopy (EM), scientists can visualize minute features of whole cells, cellular organelles, and biological assemblies (Li et al, 2009; Hu et al, 2015; Oikonomou & Jensen, 2016; Deng et al, 2017; Murata & Wolf, 2018; Varano et al, 2019). Improvements in electron optics, direct detectors, computing algorithms, and molecular modeling software contributed to this revolutionary capability (Bammes et al, 2012; Migunov et al, 2015). To uncover nanoscale information among flexible molecules, cryo-EM samples are pristinely preserved in thin layers of vitreous ice. To uncover nanoscale information among flexible molecules, cryo-EM samples are pristinely preserved in thin layers of vitreous ice This preservation step yields individual snapshots of moving parts, instantly frozen in time. One can begin to extrapolate nanoscale mechanics in a time-resolved manner (Penczek et al, 1994; Frank et al, 1995; Frank, 2006; Shi et al, 2009)
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