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.

Oxygen-evolving complex of photosystem II (PSII) is a tetramanganese calcium penta-oxygenic cluster (Mn 4 CaO 5 ) catalyzing light-induced water oxidation through several intermediate states (S-states) by a mechanism that is not fully understood. To elucidate the roles of Ca 2+ in this cluster and the possible location of water substrates in this process, we crystallized Sr 2+ -substituted PSII from Thermosynechococcus vulcanus, analyzed its crystal structure at a resolution of 2.1 Å, and compared it with the 1.9 Å structure of native PSII. Our analysis showed that the position of Sr was moved toward the outside of the cubane structure of the Mn 4 CaO 5 -cluster relative to that of Ca 2+ , resulting in a general elongation of the bond distances between Sr and its surrounding atoms compared with the corresponding distances in the Ca-containing cluster. In particular, we identified an apparent elongation in the bond distance between Sr and one of the two terminal water ligands of Ca 2+ , W3, whereas that of the Sr-W4 distance was not much changed. This result may contribute to the decrease of oxygen evolution upon Sr 2+ -substitution, and suggests a weak binding and rather mobile nature of this particular water molecule (W3), which in turn implies the possible involvement of this water molecule as a substrate in the O-O bond formation. In addition, the PsbY subunit, which was absent in the 1.9 Å structure of native PSII, was found in the Sr-PSII structure.membrane protein | water-splitting | structural changes | cyanobacteria | artificial photosynthesis P hotosynthetic light-induced water-splitting produces electrons, protons, and molecular oxygen from water; the latter product maintains the oxygenic atmosphere indispensable for sustaining oxygenic life on the earth. This process takes place in photosystem II (PSII), a multisubunit membrane protein complex containing 20 subunits with an overall molecular mass of 350 kDa (1, 2). In PSII, light is absorbed by the reaction center chlorophyll a molecules (P680), which initiates a series of electron transfer reactions leading to the formation of the charge separated state P 680 + /Q A − (the first quinone acceptor of PSII). The oxidized reaction center chlorophyll is rapidly reduced by a redox active tyrosine (Y Z ), which is the Tyr161 residue of the D1 protein. Subsequently, the oxidized Y Z abstracts an electron from an oxygen-evolving complex (OEC), which is located close to Y Z . Upon abstraction of four electrons from OEC, two water molecules are split into protons and molecular oxygen. Thus, the structure of OEC cycles through five distinct states termed S i (where i = 0-4), with the S 0 -state being the most reduced one, and the S 4 -state a transit one. Among these S-states, the S 1 -state is dark stable, and the molecular oxygen is produced in the transition of S 3 -(S 4 )-S 0 (3).To understand the mechanism of photo-induced water-splitting, extensive studies have been carried out to reveal the structure of OEC (4-10). From these studies, it has been clear th...

The European XFEL (EuXFEL) is a 3.4-km long X-ray source, which produces femtosecond, ultrabrilliant and spatially coherent X-ray pulses at megahertz (MHz) repetition rates. This X-ray source has been designed to enable the observation of ultrafast processes with near-atomic spatial resolution. Time-resolved crystallographic investigations on biological macromolecules belong to an important class of experiments that explore fundamental and functional structural displacements in these molecules. Due to the unusual MHz X-ray pulse structure at the EuXFEL, these experiments are challenging. Here, we demonstrate how a biological reaction can be followed on ultrafast timescales at the EuXFEL. We investigate the picosecond time range in the photocycle of photoactive yellow protein (PYP) with