Abstract
Serial crystallography (SX) provides an opportunity to observe the molecular dynamics of macromolecular structures at room temperature via pump-probe studies. The delivery of crystals embedded in a viscous medium via an injector or syringe is widely performed in synchrotrons or X-ray free-electron laser facilities with low repetition rates. Various viscous media have been developed; however, there are cases in which the delivery material undesirably interacts chemically or biologically with specific protein samples, or changes the stability of the injection stream, depending on the crystallization solution. Therefore, continued discovery and characterization of new delivery media is necessary for expanding future SX applications. Here, the preparation and characterization of new polysaccharide (wheat starch (WS) and alginate)-based sample delivery media are introduced for SX. Crystals embedded in a WS or alginate injection medium showed a stable injection stream at a flow rate of < 200 nL/min and low-level X-ray background scattering similar to other hydrogels. Using these media, serial millisecond crystallography (SMX) was performed, and the room temperature crystal structures of glucose isomerase and lysozyme were determined at 1.9–2.0 Å resolutions. WS and alginate will allow an expanded application of sample delivery media in SX experiments.
Highlights
Traditional X-ray crystallography plays an important role in structural biology, as it allows detailed understanding of chemical and biological functions and, the mechanisms underlying them [1,2]
It was expected that wheat starch (WS) and alginate could be used as delivery materials in gel or high-viscosity states
The preparation and characterization of WS and alginate delivery media were reported for SX experiments
Summary
Traditional X-ray crystallography plays an important role in structural biology, as it allows detailed understanding of chemical and biological functions and, the mechanisms underlying them [1,2]. Alginates have an almost temperature-independent sol/gel transition in the presence of multivalent cations (e.g., Ca2+), which is applicable in the immobilization of living cells [43] Since these materials have oligosaccharides with different chemical structures than those previously reported, they can provide an expanded opportunity for use as a delivery medium for sugar-binding protein crystals in SX experiments. The preparation and characterization of wheat starch (WS) and alginate as sample delivery media are introduced for SX experiment Both delivery media produced stable injection streams at flow rates
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