Abstract
X-ray crystallographic methods can be used to visualize macromolecules at high resolution. This provides an understanding of molecular mechanisms and an insight into drug development and rational engineering of enzymes used in the industry. Although conventional synchrotron-based X-ray crystallography remains a powerful tool for understanding molecular function, it has experimental limitations, including radiation damage, cryogenic temperature, and static structural information. Serial femtosecond crystallography (SFX) using X-ray free electron laser (XFEL) and serial millisecond crystallography (SMX) using synchrotron X-ray have recently gained attention as research methods for visualizing macromolecules at room temperature without causing or reducing radiation damage, respectively. These techniques provide more biologically relevant structures than traditional X-ray crystallography at cryogenic temperatures using a single crystal. Serial femtosecond crystallography techniques visualize the dynamics of macromolecules through time-resolved experiments. In serial crystallography (SX), one of the most important aspects is the delivery of crystal samples efficiently, reliably, and continuously to an X-ray interaction point. A viscous delivery medium, such as a carrier matrix, dramatically reduces sample consumption, contributing to the success of SX experiments. This review discusses the preparation and criteria for the selection and development of a sample delivery medium and its application for SX.
Highlights
The field of structural biology using synchrotron radiation has provided an understanding of the functional molecular mechanisms of macromolecules such as proteins and nucleic acids [1,2,3]
After the development of Lipidic cubic phase (LCP) injection medium, other sample delivery media such as mineral-oil based grease [52], Vaseline [53], agarose [54], hyaluronic acid (HA) [55], synthetic grease [55], hydroxyethyl cellulose (HEC) [56], nuclear grade grease [56], carboxymethyl cellulose sodium salt (NaCMC) [57], Pluronic F-127 (F-127) [57], poly(ethylene oxide) (PEO) [58], and polyacrylamide (PAM) [59] as the carrier matrix have been applied in SFX or SMX experiments
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Summary
The field of structural biology using synchrotron radiation has provided an understanding of the functional molecular mechanisms of macromolecules such as proteins and nucleic acids [1,2,3]. After the development of LCP injection medium, other sample delivery media such as mineral-oil based grease [52], Vaseline (petroleum jelly) [53], agarose [54], hyaluronic acid (HA) [55], synthetic grease [55], hydroxyethyl cellulose (HEC) [56], nuclear grade grease [56], carboxymethyl cellulose sodium salt (NaCMC) [57], Pluronic F-127 (F-127) [57], poly(ethylene oxide) (PEO) [58], and polyacrylamide (PAM) [59] as the carrier matrix have been applied in SFX or SMX experiments. These two syringes were connected using a coupler (Figure 4B), mixed until the crystal suspension was uniformly distributed in the delivery medium (Figure 4C) This mixture was further transferred to the sample injector and used in the SFX experiment (Figure 4D). Mebio, guar, xanthan, guar, and tragacanth materials have been suggested as possible delivery materials [53,57], but sufficient information has not been reported and these have been excluded from this review
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