@article {68010, title = {In situ single particle classification reveals distinct 60S maturation intermediates in cells.}, journal = {eLife}, year = {2022}, month = {2022 Aug 25}, abstract = {

Electron cryo-microscopy (cryo-EM) can generate high-resolution views of cells with faithful preservation of molecular structure.\ In situ\ cryo-EM, therefore, has enormous potential to reveal the atomic details of biological processes in their native context. However, in practice, the utility of\ in situ\ cryo-EM is limited by the difficulty of reliably locating and confidently identifying molecular targets (particles) and their conformational states in the crowded cellular environment. We recently showed that 2DTM, a fine-grained template-based search applied to cryo-EM micrographs, can localize particles in two-dimensional views of cells with high precision. Here we demonstrate that the signal-to-noise ratio (SNR) observed with 2DTM can be used to differentiate related complexes in focused ion beam (FIB)-milled cell sections. We apply this method in two contexts to locate and classify related intermediate states of 60S ribosome biogenesis in the\ Saccharomyces cerevisiae\ cell nucleus. In the first, we separate the nuclear pre-60S population from the cytoplasmic mature 60S population, using the subcellular localization to validate assignment. In the second, we show that relative 2DTM SNRs can be used to separate mixed populations of nuclear pre-60S that are not visually separable. We use a maximum likelihood approach to define the probability of each particle belonging to each class, thereby establishing a statistic to describe the confidence of our classification. Without the need to generate 3D reconstructions, 2DTM can be applied even when only a few target particles exist in a cell.

}, doi = {10.7554/eLife.79272}, url = {https://doi.org/10.7554/eLife.79272}, author = {Bronwyn A. Lucas and Kexin Zhang and Sarah Loerch and Nikolaus Grigorieff} } @article {67872, title = {Small-molecule ligands can inhibit -1 programmed ribosomal frameshifting in a broad spectrum of coronaviruses.}, journal = {bioRxiv}, year = {2021}, month = {2021 Aug 06}, abstract = {

Recurrent outbreaks of novel zoonotic coronavirus (CoV) diseases since 2000 have high-lighted the importance of developing therapeutics with broad-spectrum activity against CoVs. Because all CoVs use -1 programmed ribosomal frameshifting (-1 PRF) to control expression of key viral proteins, the frameshift signal in viral mRNA that stimulates -1 PRF provides a promising potential target for such therapeutics. To test the viability of this strategy, we explored a group of 6 small-molecule ligands, evaluating their activity against the frameshift signals from a panel of representative bat CoVs{\textemdash}the most likely source of future zoonoses{\textemdash}as well as SARS-CoV-2 and MERS-CoV. We found that whereas some ligands had notable activity against only a few of the frameshift signals, the serine protease inhibitor nafamostat suppressed -1 PRF significantly in several of them, while having limited to no effect on -1 PRF caused by frameshift signals from other viruses used as negative controls. These results suggest it is possible to find small-molecule ligands that inhibit -1 PRF specifically in a broad spectrum of CoVs, establishing the frameshift signal as a viable target for developing pan-coronaviral therapeutics.

}, doi = {10.1101/2021.08.06.455424}, url = {https://doi.org/10.1101/2021.08.06.455424}, author = {Sneha Munshi and Krishna Neupane and Sandaru M. Ileperuma and Matthew T.J. Halma and Jamie A. Kelly and Clarissa F. Halpern and Jonathan D. Dinman and Sarah Loerch and Michael T. Woodside} }