Development of a diffractometer specialized for electron diffraction experiments.

Along with the adoption of three-dimensional electron diffraction (3D ED/MicroED) as a mainstream tool for structure determination from sub-micron single crystals, questions about best practices regarding each step along the workflow, from data collection to structure solutions, arise. In this paper, we discuss three particular aspects of a 3D ED/MicroED experiment which, after hundreds of structures solved in Rigaku’s laboratories, we have found to be important to consider carefully. First, for a representative model system of a hydrated compound (trehalose dihydrate), we show that cryo-transfer of the sample into the diffractometer is an effective means to prevent dehydration, while cooling of the sample without cryo-transfer yields a marginal improvement only. Next, we demonstrate for a small (tyrosine) and a large (clarithromycin) organic compound, how a simplified and fast workflow for dynamical diffraction calculations can determine absolute crystal structures with high confidence. Finally, we discuss considerations and trade-offs for choosing an optimal effective crystal-to-detector distance; while a long distance is mandatory for a protein (thaumatin) example, even a small molecule with difficult diffraction behavior (cystine) yields superior results at longer distances than the one used by default.

Prenylated indole alkaloids featuring spirooxindole rings possess a 3R or 3S carbon stereocenter, which determines the bioactivities of these compounds. Despite the stereoselective advantages of spirooxindole biosynthesis compared with those of organic synthesis, the biocatalytic mechanism for controlling the 3R or 3S-spirooxindole formation has been elusive. Here, we report an oxygenase/semipinacolase CtdE that specifies the 3S-spirooxindole construction in the biosynthesis of 21R-citrinadin A. High-resolution X-ray crystal structures of CtdE with the substrate and cofactor, together with site-directed mutagenesis and computational studies, illustrate the catalytic mechanisms for the possible β-face epoxidation followed by a regioselective collapse of the epoxide intermediate, which triggers semipinacol rearrangement to form the 3S-spirooxindole. Comparing CtdE with PhqK, which catalyzes the formation of the 3R-spirooxindole, we reveal an evolutionary branch of CtdE in specific 3S spirocyclization. Our study provides deeper insights into the stereoselective catalytic machinery, which is important for the biocatalysis design to synthesize spirooxindole pharmaceuticals.

The performance of the new 52 kHz frame rate Rigaku XSPA-500k detector was characterized on beamline 8-ID-I at the Advanced Photon Source at Argonne for X-ray photon correlation spectroscopy (XPCS) applications. Due to the large data flow produced by this detector (0.2 PB of data per 24 h of continuous operation), a workflow system was deployed that uses the Advanced Photon Source data-management (DM) system and high-performance software to rapidly reduce area-detector data to multi-tau and two-time correlation functions in near real time, providing human-in-the-loop feedback to experimenters. The utility and performance of the workflow system are demonstrated via its application to a variety of small-angle XPCS measurements acquired from different detectors in different XPCS measurement modalities. The XSPA-500k detector, the software and the DM workflow system allow for the efficient acquisition and reduction of up to ∼10⁹ area-detector data frames per day, facilitating the application of XPCS to measuring samples with weak scattering and fast dynamics.