Shortening injection matrix for serial crystallography

Area of Science:

• Structural Biology
• Biophysics
• Crystallography

Background:

• Serial crystallography requires continuous sample delivery for room-temperature structure determination.
• Viscous hydrophobic media, such as lipidic cubic phase (LCP), are commonly used for sample delivery in serial X-ray crystallography (SX).
• Developing new hydrophobic media can enhance SX capabilities for diverse samples.

Purpose of the Study:

• To introduce and evaluate fat-based shortening as a novel hydrophobic delivery medium for SX experiments.
• To assess the handling, flow characteristics, and X-ray scattering properties of shortening.
• To demonstrate the utility of shortening for room-temperature crystal structure determination via SX.

Main Methods:

• Fat-based shortening was tested as a viscous delivery medium for SX.
• Shortening's flow rate stability was assessed using a syringe at low flow rates (<200 nl/min).
• X-ray scattering of shortening was analyzed, and serial millisecond crystallography (SMC) was performed on lysozyme and glucose isomerase crystals delivered in shortening.

Main Results:

• Shortening provided a stable injection stream at low flow rates, comparable to LCP.
• X-ray exposure of shortening generated minimal background scattering, with intensities similar to or lower than LCP.
• Room-temperature crystal structures of lysozyme and glucose isomerase were successfully determined at 1.5-2.0 Å resolution using shortening as the delivery medium.

Conclusions:

• Fat-based shortening is a viable, low-cost, and easy-to-handle alternative hydrophobic delivery medium for SX.
• Shortening facilitates efficient sample delivery and enables high-resolution structure determination in serial crystallography.
• This finding expands the toolkit for sample delivery in X-ray scattering studies.