Beef tallow injection matrix for serial crystallography

Abstract

Serial crystallography (SX) enables the visualization of the time-resolved molecular dynamics of macromolecular structures at room temperature while minimizing radiation damage. In SX experiments, the delivery of a large number of crystals into an X-ray interaction point in a serial and stable manner is key. Sample delivery using viscous medium maintains the stable injection stream at low flow rates, markedly reducing sample consumption compared with that of a liquid jet injector and is widely applied in SX experiments with low repetition rates. As the sample properties and experimental environment can affect the stability of the injection stream of a viscous medium, it is important to develop sample delivery media with various characteristics to optimize the experimental environment. In this study, a beef tallow injection matrix possessing a higher melting temperature than previously reported fat-based shortening and lard media was introduced as a sample delivery medium and applied to SX. Beef tallow was prepared by heat treating fats from cattle, followed by the removal of soluble impurities from the extract by phase separation. Beef tallow exhibited a very stable injection stream at room temperature and a flow rate of < 10 nL/min. The room-temperature structures of lysozyme and glucose isomerase embedded in beef tallow were successfully determined at 1.55 and 1.60 Å, respectively. The background scattering of beef tallow was higher than that of previously reported fat-based shortening and lard media but negligible for data processing. In conclusion, the beef tallow matrix can be employed for sample delivery in SX experiments conducted at temperatures exceeding room temperature.

Figure 1

Beef tallow injection stream with embedded lysozyme crystals at flow rates of (A) 200 nL/min and (B) 10 nL/min. The inner diameters of the syringe needle and the injection stream of the beef tallow were 168 and < 200 μm, respectively.

Figure 2

Quality of dataset for (A) lysozyme (indexed patterns: 23,090) and (B) glucose isomerase (indexed patterns: 7976) delivered in beef tallow. CC (blue circle) and signal-to-noise ratio (red diamond) were plotted as a function of resolution.

Figure 3

Electron density map of room-temperature lysozyme embedded in beef tallow. (A) 2mFo-DFc electron density map (grey mesh, 1.2σ) of lysozyme (B) 2mFo-DFc (grey mesh, 1.2σ) and mFo-DFc (green, 3σ; red, − 3σ) electron density map around disulfide bonds of lysozyme.

Figure 4

Electron density map of room-temperature glucose isomerase embedded in beef tallow. (A) 2 mFo-DFc electron density map (grey mesh, 1.2σ) of glucose isomerase. (B) 2mFo-DFc (grey mesh, 1.2σ) and mFo-DFc (green, 3σ; red, − 3σ) electron density map of metal binding sites at the active site of glucose isomerase.

Figure 5

Analysis of X-ray background scattering of beef tallow. (A) (Left) Typical scattering image of 80% (v/v) beef tallow. Diagonals in the image on the left are used to generate the intensity plots in (B). (Right) Magnification of background scattering of beef tallow from the center of the detector to 3.0 Å in the image on the left. (B) Two-dimensional profile of the average scattering intensities of 80% (v/v) beef tallow, 80% (v/v) lard, 80% (v/v) shortening, and 60% (v/v) LCP (monoolein). Inset shows a magnified view of the intensity of background scattering in the 5–2.8 Å resolution range.