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Serial crystallography (SX) enables the determination of the structure of macromolecules or small molecules with minimal radiation damage. In particular, biomolecule structures determined using the SX technique have the advantage of providing room-temperature crystal structures with high biological relevance. The SX technique requires numerous crystals to be collected to complete three-dimensional structural information. To minimize crystal sample consumption, we introduced SX data collection with fixed-target (FT) pink-beam serial synchrotron crystallography (SSX) at the 1C beamline of Pohang Light Source II. A new sample holder consisting of a magnetic frame with a nylon mesh was developed for easy sample handling. The FT-pink-SSX diffraction data were collected by continuously scanning X-rays using a stepping motor. The room-temperature structures of glucose isomerase and lysozyme were successfully determined at a resolution of 1.7 and 2.2 Å, respectively. The use of pink-beam FT-SSX in experimental applications and data acquisition for large beam sizes is discussed. Our results provide useful information for future pink-beam SSX and SX data collection using large X-ray beams. [ABSTRACT FROM AUTHOR]

Human lysozyme is a natural non-specific immune protein that participates in the immune response of infants against bacterial and viral infections. Lysozyme is a well-known hydrolase that cleaves peptidoglycan in bacterial cell walls. Several crystal structures of human lysozyme have been reported, but little is known regarding how it recognizes sugar molecules. In this study, the crystal structures of human lysozyme in its native and two N-acetyl-α-d-glucosamine (α-D-NAG)-bound forms were determined at 1.3 Å and 1.55/1.60 Å resolution, respectively. Human lysozyme formed a typical c-type lysozyme fold and the α-D-NAG molecule was bound to the middle of subsites C and D. The N-acetyl and glucosamine groups of α-D-NAG were stabilized by hydrophobic interactions (Val117, Ala126, and Trp127), hydrogen bonds (Asn64, Asn78, Ala126, and Val128), and water bridges. Conformational changes of Arg80, Tyr81, Val128, and Arg131 of human lysozyme were observed due to the interactions of α-D-NAG with the active-site cleft. The binding configuration of α-D-NAG in human lysozyme was distinct compared with that of other sugar-bound lysozymes. Findings from this structural analysis provide a better understanding of the sugar recognition of human lysozyme during the immune response to microbial pathogens. [ABSTRACT FROM AUTHOR]

Serial crystallography (SX) enables the visualization of the time-resolved molecular dynamics of macromolecular structures at room temperature while minimizing radiation damage. In SX experiments, the delivery of a large number of crystals into an X-ray interaction point in a serial and stable manner is key. Sample delivery using viscous medium maintains the stable injection stream at low flow rates, markedly reducing sample consumption compared with that of a liquid jet injector and is widely applied in SX experiments with low repetition rates. As the sample properties and experimental environment can affect the stability of the injection stream of a viscous medium, it is important to develop sample delivery media with various characteristics to optimize the experimental environment. In this study, a beef tallow injection matrix possessing a higher melting temperature than previously reported fat-based shortening and lard media was introduced as a sample delivery medium and applied to SX. Beef tallow was prepared by heat treating fats from cattle, followed by the removal of soluble impurities from the extract by phase separation. Beef tallow exhibited a very stable injection stream at room temperature and a flow rate of < 10 nL/min. The room-temperature structures of lysozyme and glucose isomerase embedded in beef tallow were successfully determined at 1.55 and 1.60 Å, respectively. The background scattering of beef tallow was higher than that of previously reported fat-based shortening and lard media but negligible for data processing. In conclusion, the beef tallow matrix can be employed for sample delivery in SX experiments conducted at temperatures exceeding room temperature. [ABSTRACT FROM AUTHOR]

Serial crystallography (SX) using X-ray free-electron lasers or synchrotron X-rays is an emerging technique for determining the room-temperature structure of macro- and small-molecules [[9]]. Molecular dynamics are observed in all molecules, from small molecules to macromolecules, and provide information about their intrinsic structure in a spatiotemporal context within a specific environment. All natural molecules have their own physical, chemical, or biological properties and functions. [Extracted from the article]