Viscous medium-based crystal support in sample holder for fixed-target serial femtosecond crystallography

Abstract

SynopsisWe developed a viscous medium-based crystal support in a sample holder for fixed-target serial crystallography. Abstract Serial femtosecond crystallography (SFX) enables the determination of a roomtemperature crystal structure of macromolecules without causing radiation damage, as well as provides time-resolved molecular dynamics data in pump-probe experiments. Fixed-target SFX (FT-SFX) can minimize sample consumption and physical effects to crystals during sample delivery. Various types of sample holders have been developed and applied in FT-SFX; however, no sample holder has been developed that can universally mount crystals of various sizes and shapes. Here, we introduce a viscous media-based crystal support in a sample holder for FT-SFX. Crystal samples were embedded in viscous media such as gelatin and agarose, which were enclosed in a polyimide film. In the vertically placed sample holder, the viscous medium stably supported crystals between the two polyimide films without 2 crystal sinking due to gravity. Using this method, we performed FT-SFX experiments with glucose isomerase and lysozyme embedded in gelatin and agarose, respectively. The room-temperature crystal structures of glucose isomerase and lysozyme were successfully determined at 1.75 and 1.80 Å resolutions, respectively. Viscous media used in this experiment showed negligible background scattering in data processing. This method is useful for delivering crystal samples of various sizes and shapes in FT-SFX experiments.

Room-Temperature Structure of Xylitol-Bound Glucose Isomerase by Serial Crystallography: Xylitol Binding in the M1 Site Induces Release of Metal Bound in the M2 Site

“…GI has a catalytic domain comprising a (β/α) 8 -barrel, as a TIM-barrel fold, and further forms a tetrameric assembly [ 9 , 10 , 11 , 12 , 13 ]. Bivalent cations, such as Mg 2+ , Co 2+ , or Mn 2+ , are essentially required for the isomerase activity of GI [ 1 , 14 ].…”

Polyimide mesh-based sample holder with irregular crystal mounting holes for fixed-target serial crystallography

“…To obtain crystals in various orientations within the sample holder, a method for supporting crystals embedded in a viscous material between two films was applied for FT-SFX 27 . In this method, lysozyme and glucose isomerase crystals were embedded in agarose and gelatin, respectively, and placed at a thickness of 350 µm between polyimide films 27 . Although this method was effective, it may be difficult to apply when the crystals are physically damaged during the mixing step with viscous medium 27 .…”

“…In this method, lysozyme and glucose isomerase crystals were embedded in agarose and gelatin, respectively, and placed at a thickness of 350 µm between polyimide films 27 . Although this method was effective, it may be difficult to apply when the crystals are physically damaged during the mixing step with viscous medium 27 . On the contrary, FT-SSX with oscillation has been developed to collect the complete dataset by introducing oscillations in the sample holder for a short time when the fixed sample is exposed to X-rays 36 .…”