Guanosine hydrogel as a new injection matrix for protein serial X-ray crystallography

Serial X-ray crystallography is the method of choice for protein structural studies at X-ray free-electron lasers and is increasingly widely used at third- and fourth-generation synchrotron facilities. Sample delivery is a critical step in serial crystallography experiments, and the definition and control of delivery methods is especially important for time-resolved studies. Extruding protein crystals embedded in viscous matrices as a thin, stable jet is a delivery method that ensures low sample consumption. However, it requires specific characteristics, such as a constant speed, chemical compatibility and, for laser-triggered dynamic studies, optical transparency. Here, we present a new transparent carrier matrix for protein serial crystallography, based on a guanosine hydrogel. Crystal stability in the hydrogel, in terms of morphology, size and diffraction quality, along with optical properties, injection characteristics and X-ray background were tested at the ESRF synchrotron and compared with other commonly used matrices. Additionally, we present a high-quality serial femtosecond crystallography dataset collected at the Spring-8 Angstrom Compact Free-Electron Laser (Japan) using hen egg-white lysozyme microcrystals embedded in the hydrogel. The results establish that this new injection matrix is a suitable alternative to existing carriers. The main advantage is its stability, so that the composition of the aqueous phase inside the hydrogel can be easily modified and adapted to the crystallization conditions of the embedded crystals.