Structure determination of proteins and other macromolecules has historically required the growth of high-quality crystals sufficiently large to diffract x-rays efficiently while withstanding radiation damage. We applied serial femtosecond crystallography (SFX) using an x-ray free-electron laser (XFEL) to obtain high-resolution structural information from microcrystals (less than 1 micrometer by 1 micrometer by 3 micrometers) of the well-characterized model protein lysozyme. The agreement with synchrotron data demonstrates the immediate relevance of SFX for analyzing the structure of the large group of difficult-to-crystallize molecules.

The Trypanosoma brucei cysteine protease cathepsin B (TbCatB), which is involved in host protein degradation, is a promising target to develop new treatments against sleeping sickness, a fatal disease caused by this protozoan parasite. The structure of the mature, active form of TbCatB has so far not provided sufficient information for the design of a safe and specific drug against T. brucei. By combining two recent innovations, in vivo crystallization and serial femtosecond crystallography, we obtained the room-temperature 2.1 angstrom resolution structure of the fully glycosylated precursor complex of TbCatB. The structure reveals the mechanism of native TbCatB inhibition and demonstrates that new biomolecular information can be obtained by the “diffraction-before-destruction” approach of x-ray free-electron lasers from hundreds of thousands of individual microcrystals.

Protein crystallization in cells has been observed several times in nature. However, owing to their small size these crystals have not yet been used for X-ray crystallographic analysis. We prepared nano-sized in vivo–grown crystals of Trypanosoma brucei enzymes and applied the emerging method of free-electron laser-based serial femtosecond crystallography to record interpretable diffraction data. This combined approach will open new opportunities in structural systems biology.

The structure solution of T. brucei cathepsin B from 80 in vivo grown crystals with an average volume of 9 µm3 obtained by serial synchrotron crystallography at a microfocus beamline is reported.

Severe acute respiratory syndrome coronavirus is the causative agent of a respiratory disease with a high case fatality rate. During the formation of the coronaviral replication/ transcription complex, essential steps include processing of the conserved polyprotein nsp7-10 region by the main protease M pro and subsequent complex formation of the released nsp's. Here, we analyzed processing of the coronavirus nsp7-10 region using native mass spectrometry showing consumption of substrate, rise and fall of intermediate products and complexation. Importantly, there is a clear order of cleavage efficiencies, which is influenced by the polyprotein tertiary structure. Furthermore, the predominant product is an nsp7+8(2 : 2) hetero-tetramer with nsp8 scaffold. In conclusion, native MS, opposed to other methods, can expose the processing dynamics of viral polyproteins and the landscape of protein interactions in one set of experiments. Thereby, new insights into protein interactions, essential for generation of viral progeny, were provided, with relevance for development of antivirals.

Background: The causal relationship between Tau hyperphosphorylation and aggregation in neuropathology is still under debate.Results: Tau highly phosphorylated in cells increases oligomerization without pronounced aggregation. Oligomers cause reduction of dendritic spines but not cell death.Conclusion: Hyperphosphorylation does not drive Tau fibrillization but contributes to synaptotoxicity.Significance: Pathways and effects of Tau hyperphosphorylation are distinct from those of aggregation.

The new European X-ray Free-Electron Laser is the first X-ray free-electron laser capable of delivering X-ray pulses with a megahertz inter-pulse spacing, more than four orders of magnitude higher than previously possible. However, to date, it has been unclear whether it would indeed be possible to measure high-quality diffraction data at megahertz pulse repetition rates. Here, we show that high-quality structures can indeed be obtained using currently available operating conditions at the European XFEL. We present two complete data sets, one from the well-known model system lysozyme and the other from a so far unknown complex of a β-lactamase from K. pneumoniae involved in antibiotic resistance. This result opens up megahertz serial femtosecond crystallography (SFX) as a tool for reliable structure determination, substrate screening and the efficient measurement of the evolution and dynamics of molecular structures using megahertz repetition rate pulses available at this new class of X-ray laser source.

The genome of coronaviruses, including SARS‐CoV‐2, encodes for two proteases, a papain like (PLpro) protease and the so‐called main protease (Mpro), a chymotrypsin‐like cysteine protease, also named 3CLpro or non‐structural protein 5 (nsp5). Mpro is activated by autoproteolysis and is the main protease responsible for cutting the viral polyprotein into functional units. Aside from this, it is described that Mpro proteases are also capable of processing host proteins, including those involved in the host innate immune response. To identify substrates of the three main proteases from SARS‐CoV, SARS‐CoV‐2, and hCoV‐NL63 coronviruses, an LC‐MS based N‐terminomics in vitro analysis is performed using recombinantly expressed proteases and lung epithelial and endothelial cell lysates as substrate pools. For SARS‐CoV‐2 Mpro, 445 cleavage events from more than 300 proteins are identified, while 151 and 331 Mpro derived cleavage events are identified for SARS‐CoV and hCoV‐NL63, respectively. These data enable to better understand the cleavage site specificity of the viral proteases and will help to identify novel substrates in vivo. All data are available via ProteomeXchange with identifier PXD021406.