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.