Single-molecule studies can be used to study biological processes directly and in real-time. In particular, the fluorescence energy transfer between reporter dye molecules attached to specific sites on macromolecular complexes can be used to infer distance information. When several measurements are combined, the information can be used to determine the position and conformation of certain domains with respect to the complex. However, data analysis schemes that include all experimental uncertainties are highly complex, and the outcome depends on assumptions about the state of the dye molecules. Here, we present a new analysis algorithm using Bayesian parameter estimation based on Markov Chain Monte Carlo sampling and parallel tempering termed Fast-NPS that can analyse large smFRET networks in a relatively short time and yields the position of the dye molecules together with their respective uncertainties. Moreover, we show what effects different assumptions about the dye molecules have on the outcome. We discuss the possibilities and pitfalls in structure determination based on smFRET using experimental data for an archaeal transcription pre-initiation complex, whose architecture has recently been unravelled by smFRET measurements.

In electron cryomicroscopy (cryo-EM), molecular images of vitrified biological samples are obtained by conventional transmission microscopy (CTEM) using large underfocuses and subsequently computationally combined into a high-resolution three-dimensional structure. Here, we apply scanning transmission electron microscopy (STEM) using the integrated differential phase contrast mode also known as iDPC–STEM to two cryo-EM test specimens, keyhole limpet hemocyanin (KLH) and tobacco mosaic virus (TMV). The micrographs show complete contrast transfer to high resolution and enable the cryo-EM structure determination for KLH at 6.5 Å resolution, as well as for TMV at 3.5 Å resolution using single-particle reconstruction methods, which share identical features with maps obtained by CTEM of a previously acquired same-sized TMV data set. These data show that STEM imaging in general, and in particular the iDPC–STEM approach, can be applied to vitrified single-particle specimens to determine near-atomic resolution cryo-EM structures of biological macromolecules.

In the drug development process, optimization of properties and biological activities of small molecules is an important task to obtain drug candidates with optimal efficacy when first applied in subsequent clinical studies. However, despite its importance, large-scale investigations of the optimization process in early drug discovery are lacking, likely due to the absence of historical records of different chemical series used in past projects. Here, we report a retrospective reconstruction of ∼3000 chemical series from the Novartis compound database, which allows us to characterize the general properties of chemical series as well as the time evolution of structural properties, ADMET properties, and target activities. Our data-driven approach allows us to substantiate common MedChem knowledge. We find that size, fraction of sp 3 -hybridized carbon atoms (F sp 3 ), and the density of stereocenters tend to increase during optimization, while the aromaticity of the compounds decreases. On the ADMET side, solubility tends to increase and permeability decreases, while safety-related properties tend to improve. Importantly, while ligand efficiency decreases due to molecular growth over time, target activities and lipophilic efficiency tend to improve. This emphasizes the heavy-atom count and log D as important parameters to monitor, especially as we further show that the decrease in permeability can be explained with the increase in molecular size. We highlight overlaps, shortcomings, and differences of the computationally reconstructed chemical series compared to the series used in recent internal drug discovery projects and investigate the relation to historical projects.

Stable capsid structures of viruses protect viral RNA while they also require controlled disassembly for releasing the viral genome in the host cell. A detailed understanding of viral disassembly processes and the involved structural switches is still lacking. This process has been extensively studied using tobacco mosaic virus (TMV), and carboxylate interactions are assumed to play a critical part in this process. Here, we present two cryo‐EM structures of the helical TMV assembly at 2.0 and 1.9 Å resolution in conditions of high Ca2+ concentration at low pH and in water. Based on our atomic models, we identify the conformational details of the disassembly switch mechanism: In high Ca2+/acidic pH environment, the virion is stabilized between neighboring subunits through carboxyl groups E95 and E97 in close proximity to a Ca2+ binding site that is shared between two subunits. Upon increase in pH and lower Ca2+ levels, mutual repulsion of the E95/E97 pair and Ca2+ removal destabilize the network of interactions between adjacent subunits at lower radius and release the switch for viral disassembly.