Lard Injection Matrix for Serial Crystallography

Ki Hyun Nam

doi:10.3390/ijms21175977

Abstract

Serial crystallography (SX) using X-ray free electron laser or synchrotron X-ray allows for the determination of structures, at room temperature, with reduced radiation damage. Moreover, it allows for the study of structural dynamics of macromolecules using a time-resolved pump-probe, as well as mix-and-inject experiments. Delivering a crystal sample using a viscous medium decreases sample consumption by lowering the flow rate while being extruded from the injector or syringe as compared to a liquid jet injector. Since the environment of crystal samples varies, continuous development of the delivery medium is important for extended SX applications. Herein, I report the preparation and characterization of a lard-based sample delivery medium for SX. This material was obtained using heat treatment, and then the soluble impurities were removed through phase separation. The lard injection medium was highly stable and could be injected via a syringe needle extruded at room temperature with a flow rate < 200 nL/min. Serial millisecond crystallography experiments were performed using lard, and the room temperature structures of lysozyme and glucose isomerase embedded in lard at 1.75 and 1.80 Å, respectively, were determined. The lard medium showed X-ray background scattering similar or relatively lower than shortenings and lipidic cubic phase; therefore, it can be used as sample delivery medium in SX experiments.

Highlights

The serial crystallography (SX) technique allows for structure determination of a variety of materials, from macromolecules to small molecules, at room temperature [1,2,3]
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 X-ray free electron laser (XFEL) facility with low repetition rate or synchrotron [10,11,24,26,27,28,29,30,31,32,33]
Shortening materials have been previously used as a stable injection stream in SMX experiments [31], but due to issues related to their Tm values, use at room temperature was limited

Summary

Introduction

The serial crystallography (SX) technique allows for structure determination of a variety of materials, from macromolecules to small molecules, at room temperature [1,2,3]. SX, using an XFEL or synchrotron X-ray, can be used to observe structural flexibility of molecules at room temperature and of radiation-sensitive materials (such as metalloproteins), in addition to the dynamics of molecules [3,4,5,6,7]. 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 a fixed-target sample holder for SX, a viscous material can hold the crystal samples of various shapes and sizes between two films [25]

Methods

Results

Conclusion