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.
Highlights
Background scattering analysisShortening A, shortening B, and lipidic cubic phase (LCP) delivery media were passed through a syringe needle with a 168 μm inner diameter (ID)
To apply shortening to an injection matrix for an SX experiment, the melting temperatures of shortenings A and B were screened in the range of 20 to 40 °C
Because the melting temperature of the shortening is below typical body temperature, the liquid phase of the shortening can be achieved by holding the glass vial containing the shortening by hand, but this takes several minutes
Summary
Shortening A, shortening B, and LCP delivery media were passed through a syringe needle with a 168 μm ID. Each delivery medium was exposed to X-rays of 1.3 × 1012 photons/ flux for 100 ms. The background scattering from 1.6 Å to the beam centre was analysed using the average intensity for 100 images. The background intensities of the delivery media were analysed using ADXV The coordinates and structure factors have been deposited in the Protein Data Bank under the accession code 6KCA (Glucose isomerase-shortening A), 6KCB (Lysozyme-shortening A), 6KCC (Glucose isomerase-shortening B), 6KCD (Lysozyme-shortening B). Diffraction images have been deposited to CXIDB under ID (glucose isomerase-shortening A), (Lysozyme-shortening A), (Glucose isomerase-shortening B) and (Lysozyme-shortening B)
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