Polysaccharide-Based Injection Matrix for Serial Crystallography

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.

Lard Injection Matrix for Serial Crystallography

“…Equal volumes (100 ul) of lysozyme solution and crystallization solution (100 μl) containing 0.1M Na-acetate, pH 4.6, 6% ( w / v ) PEG 8000, and 10% ( w / v ) NaCl were mixed in a 1.5 mL microcentrifuge tube, vortexed, and finally incubated overnight at 22 °C. Glucose isomerase, from Streptomyces rubiginosus , were purchased from Hampton Research (HR7-098, Aliso Viejo, CA, USA), which supplied the crystalline, and thus were directly used in the SX experiment without post-crystallization, as previously reported [ 31 , 32 ]. Products containing glucose isomerase crystals were stored at 4 °C for future use.…”

“…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 ].…”

“…Meanwhile, LCP medium produces certain high background scattering [ 10 ]. 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.…”

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 ].…”

Approach of Serial Crystallography

“…Moreover, a sample delivery method is required to continuously and reliably deliver a large number of crystal samples to the X-ray interaction point for diffraction data collection [34,35]. Various sample delivery methods such as the use of injectors [36,37], syringes [38,39], injection with carrier matrix [35,[37][38][39][40][41][42][43][44], fixed-target scanning [33,[45][46][47][48], microfluidics [49][50][51], and conveyor belt approaches [52,53] are developed. These techniques are used to carry out the SX experiment and crystal structure can be determined successfully.…”