The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID-19, we have performed a high-throughput X-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (Mpro), which is essential for viral replication. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to Mpro. In subsequent cell-based viral reduction assays, one peptidomimetic and six non-peptidic compounds showed antiviral activity at non-toxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.

The room-temperature structure of lysozyme is determined using 40000 individual diffraction patterns from micro-crystals flowing in liquid suspension across a synchrotron microfocus beamline.

Unravelling the interaction of biological macromolecules with ligands and substrates at high spatial and temporal resolution remains a major challenge in structural biology. The development of serial crystallography methods at X-ray free-electron lasers and subsequently at synchrotron light sources allows new approaches to tackle this challenge. Here, a new polyimide tape drive designed for mix-and-diffuse serial crystallography experiments is reported. The structure of lysozyme bound by the competitive inhibitor chitotriose was determined using this device in combination with microfluidic mixers. The electron densities obtained from mixing times of 2 and 50 s show clear binding of chitotriose to the enzyme at a high level of detail. The success of this approach shows the potential for high-throughput drug screening and even structural enzymology on short timescales at bright synchrotron light sources.

Kinases of the MARK/Par-1 family of S/T protein kinases are regulators of diverse cellular processes in Caenorhabditis elegans, Drosophila, yeast, and mammalian cells. They are involved in nematode embryogenesis, epithelial cell polarization, cell signaling, and neuronal differentiation. MARK phosphorylates microtubule-associated proteins such as tau and is a key regulator of microtubule-based intracellular transport. Hyperphosphorylation of tau causes defects in neuronal transport and may induce abnormal aggregation of tau in Alzheimer disease and other tauopathies. Recent high-resolution structure analysis of MARK fragments covering the kinase domain and accessory regulatory domains has revealed important details regarding the autoregulation of MARK, but their interpretation has remained controversial. Here we focus on the structural aspects of MARK activity and autoregulation. Comparison of the available MARK structures with related kinases of the AMPK family and with new structures of MARK isoforms (MARK2 and 3) reveals unexpected structural similarities between these kinases that may help to resolve the existing controversies.

Since 2013, three beamlines for macromolecular crystallography are available to users at the thirdgeneration synchrotron PETRA III in Hamburg: P11, P13 and P14, the latter two operated by EMBL. Beamline P11 is operated by DESY and is equipped with a Pilatus 6M detector. Together with the photon flux of 2 × 10 13 ph/s provided by the very brilliant X-ray source of PETRA III, a full data set can be typically collected in less than 2 min. P11 provides state-of-the-art microfocusing capabilities with beam sizes down to 1 × 1 µm 2 , which makes the beamline ideally suited for investigation of microcrystals and serial crystallography experiments. An automatic sample changer allows fast sample exchange in less than 20 s, which enables high-throughput crystallography and fast crystal screening. For sample preparation, an S2 biosafety laboratory is available in close proximity to the beamline.

At low emittance synchrotron sources it has become possible to perform structure determinations from the measurement of multiple microcrystals which were previously considered too small for diffraction experiments. Conventional mounting techniques do not fulfill the requirements of these new experiments. They significantly contribute to background scattering and it is difficult to locate the crystals, making them incompatible with automated serial crystallography. We have developed a micro-fabricated sample holder from single crystalline silicon with micropores, which carries up to thousands of crystals and significantly reduces the background scattering level. For loading, the suspended microcrystals are pipetted onto the chip and excess mother liquor is subsequently soaked off through the micropores. Crystals larger than the pore size are retained and arrange themselves according to the micropore pattern. Using our chip we were able to collect 1.5 Å high resolution diffraction data from protein microcrystals with sizes of 4 micrometers and smaller.

The Ser/Thr kinase MARK2 phosphorylates tau protein at sites that cause detachment from microtubules in Alzheimer neurofibrillary degeneration. Homologs of MARK2 include Par-1 in C. elegans and Drosophila, which generates embryonic polarity. We report the X-ray structure of the catalytic and ubiquitin-associated domains (UBA) of human MARK2. The activity was altered by mutations in the ATP binding site and/or activation loop. The catalytic domain shows the small and large lobes typical of kinases. The substrate cleft is in an inactive, open conformation in the inactivated and the wild-type structure. The UBA domain is attached via a taut linker to the large lobe of the kinase domain and leans against a hydrophobic patch on the small lobe. The UBA structure is unusual because the orientation of its third helix is inverted, relative to previous structures. Possible implications of the structure for the regulation of kinase activity are discussed.

A neutral C4 cumulene 1 that includes a cyclic alkyl(amino) carbene (cAAC), its air-stable radical cation 1(·+) , and dication 1(2+) have been synthesized. The redox property of 1(·+) was studied by cyclic voltammetry. EPR and theoretical calculations show that the unpaired electron in 1(·+) is mainly delocalized over the central C4 backbone. The commercially available CBr4 is utilized as a source of dicarbon in the cumulene synthesis.