Shortening injection matrix for serial crystallography

Abstract

Serial crystallography allows crystal structures to be determined 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 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 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.

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

“…The monomer subunit further forms the functional tetrameric assembly observed in the crystal symmetry ( Figure 2 b). The subunit topology and tetrameric assembly of SruGIs were identical with previously reported crystal structures of SruGI [ 10 , 11 , 13 , 29 , 30 ]. The structure of native SruGI was highly similar to other room-temperature crystal structures of native SruGI determined by serial crystallography (PDB codes: 6KCA, 6KD2, 7BVL, and 7CK0) [ 10 , 11 , 29 , 30 ], with r.m.s.d.…”

Polysaccharide-Based Injection Matrix for Serial Crystallography

“…Sample delivery media can be classified into either amphiphilic, hydrophobic, or hydrophilic materials [33]. Amphiphilic sample delivery medium, such as lipidic cubic phases (LCPs) [17], and hydrophobic sample delivery media, such as greases [19,[34][35][36] and shortening [37], produce a stable injection stream with high viscosity while exhibiting certain background scattering rings from the delivery material. Hydrophilic delivery materials can be further classified into sugar-based media, such as agarose [38], hyaluronic acid (HA) [34], hydroxyethyl cellulose (HEC) [35], and sodium carboxymethyl cellulose (NaCMC) [39], and polymer-based media, such as Pluronic F-127 [39], poly(ethylene oxide) [14], and polyacrylamide (PAM) [40].…”

Towards an Optimal Sample Delivery Method for Serial Crystallography at XFEL

“…To take advantage of this low sample consumption and broaden the applicability to samples not grown or not sufficiently stable in LCP [109], a wide variety of viscous matrixes with broad hydrophobic and hydrophilic compatibility (reviewed in [110]) have been demonstrated for serial crystallography data collection. These includes mineral oil grease [111], agarose [112], hyaluronic acid [113], synthetic grease [113], Vaseline [114], nuclear grease ( [115], hydroxyethyl cellulose [115], sodium carboxymethyl cellulose [116], pluronic F-127 [116], polyethylene oxide [117], polyacrylamide [118], fat-based shortening [119], dextrin palmitate/paraffin grease [120], and dextrin palmitate/dialkyl tetraphenyl ether grease [120]. To utilize these matrixes crystals grown in batch mode are spun down to remove excess mother liquor before embedding into the matrix by manual mixing either with a spatula or Hamilton syringe.…”

“…To utilize these matrixes crystals grown in batch mode are spun down to remove excess mother liquor before embedding into the matrix by manual mixing either with a spatula or Hamilton syringe. A more gentle method without centrifugation is allowing crystal suspensions to settle at the bottom of Hamilton syringes before excess mother liquor is pushed out from the top [119]. After mixing crystals should be checked under the microscope for signs of damage, either due to chemical incompatibility with the matrix or shear force from mechanical mixing.…”