Shortening injection matrix for serial crystallography

Abstract

Serial crystallography (SX) allows crystal structures to be observed at room temperature through the steady delivery of crystals to the X-ray interaction point. Viscous delivery media are advantageous because they afford efficient sample delivery from an injector or syringe at a low flow rate. Hydrophobic delivery media, such as lipidic cubic phase (LCP) or grease, provide a very stable injection stream and are widely used. The development of new hydrophobic delivery materials can expand opportunities for future SX studies with various samples. Here, I introduce fat-based shortening as a delivery medium for SX experiments.This material is commercially available at low cost and is straightforward to handle because its phase (i.e., solid or liquid) can be controlled by temperature. Shortening was extruded from a syringe needle in a very stable injection stream even below 200 nl/min. X-ray exposed shortening produced several background scattering rings, which have similar or lower intensities than those of LCP and contribute negligibly to data processing. Serial millisecond crystallography was performed using two shortening delivery media, and the room temperature crystal structures of lysozyme and glucose isomerase were successfully determined at resolutions of 1.5-2.0 Å. Therefore, shortening can be used as a sample delivery medium in SX experiments. more biologically reliable crystal structure of macromolecules beyond conventional X-ray crystallography using single crystal diffraction at cryogenic temperatures. In SX experiments, it is important to deliver the crystal sample serially and stably to the X-ray interaction point. 5Delivering the sample reliably not only reduces sample consumption but also enables data collection for accurate responses in time-resolved studies. 6 Among the various sample delivery methods, including liquid jet injector, 7 high viscosity injector, 8,9 fixed-target scanning, 10-12 and microfluidics, 13,14 sample delivery using a viscous medium with an injector or syringe is widely applied to serial femtosecond crystallography (SFX) or serial millisecond crystallography (SMX), which can successfully determine structure using an XFEL or synchrotron, respectivley. [15][16][17][18][19][20][21][22] The advantage of crystal delivery with a viscous delivery medium is the drastic reduction of sample consumption by lowering the flow rate of the sample loading from the injector or syringe. 22Based on their physical and chemical properties, sample delivery media can be categorized as either lipidic cubic phase (LCP), 15 oil-based, [16][17][18] or hydrogel-based. 17-21 Among them, LCP and grease, which have hydrophobic characteristics, provide the most stable injection streams and are the most widely used delivery media in SX experiments. 15-18 LCP, which is usually used with monoolein, is widely applied in the process of membrane protein crystallization, and its crystallized membrane protein embedded in LCP can be used as a sample delivery medium. 15,23 The LCP delivery medium enables the sample t...

Lard Injection Matrix for Serial Crystallography

“…Various sample delivery methods, such as injectors [ 9 , 10 ], syringes [ 11 , 12 , 13 ], capillaries [ 14 , 15 ], fixed targets [ 16 , 17 , 18 , 19 , 20 , 21 ], and microfluidics [ 22 , 23 ], have been developed and applied to SX experiments; viscous materials can be used in combination with some sample delivery systems [ 15 , 24 , 25 ]. Crystal samples enclosed in viscous materials provide a stable injection stream even at low flow rates when extruded from an injector or syringe, thereby reducing sample consumption in the XFEL facility with low repetition rate or synchrotron [ 10 , 11 , 24 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 ]. In the capillary method with viscous medium, sample consumption can be reduced by allowing the sample to be delivered at a low flow rate by preventing the crystal sample from being sunk by gravity [ 15 ].…”

“…Hydrophilic sample delivery materials, including agarose [ 26 ], hyaluronic acid [ 29 ], hydroxyethyl cellulose [ 30 ], sodium carboxymethyl cellulose [ 37 ], Pluronic F-127 [ 37 ], poly(ethylene oxide) [ 7 ], polyacrylamide [ 28 ], wheat starch [ 32 ], and alginate [ 32 ], have low background scattering compared to an LCP medium. Hydrophobic delivery materials, such as greases [ 11 , 29 , 30 , 33 ] and shortening [ 31 ], provide a stable injection stream due to viscous properties higher than other types of materials. Among them, shortening is a harmless material and therefore its injection matrix in SX is safe, easy to handle with temperature control, and has a lower cost than other delivery media [ 31 ].…”

“…Hydrophobic delivery materials, such as greases [ 11 , 29 , 30 , 33 ] and shortening [ 31 ], provide a stable injection stream due to viscous properties higher than other types of materials. Among them, shortening is a harmless material and therefore its injection matrix in SX is safe, easy to handle with temperature control, and has a lower cost than other delivery media [ 31 ]. However, although the melting temperature (Tm) of the shortening used in the experiment was higher than the room temperature, the stability of the shortening injection stream deteriorated because of heat generated from the beamline equipment during data collection in the experimental hutch [ 31 ].…”

“…Among them, shortening is a harmless material and therefore its injection matrix in SX is safe, easy to handle with temperature control, and has a lower cost than other delivery media [ 31 ]. However, although the melting temperature (Tm) of the shortening used in the experiment was higher than the room temperature, the stability of the shortening injection stream deteriorated because of heat generated from the beamline equipment during data collection in the experimental hutch [ 31 ]. Consequently, in this experiment, a stable shortening injection stream was produced when the temperature of the experimental hutch was maintained at 20 °C [ 31 ].…”

“…However, although the melting temperature (Tm) of the shortening used in the experiment was higher than the room temperature, the stability of the shortening injection stream deteriorated because of heat generated from the beamline equipment during data collection in the experimental hutch [ 31 ]. Consequently, in this experiment, a stable shortening injection stream was produced when the temperature of the experimental hutch was maintained at 20 °C [ 31 ]. Although the SX application using shortening matrix was successfully proved, this material may be of limited use when the structure of the target material is to be observed above room temperature.…”