Sample Delivery Media for Serial Crystallography

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.

Keywords: X-ray free electron laser (XFEL); carrier matrix; delivery medium; sample delivery; serial crystallography (SX); serial femtosecond crystallography (SFX); serial millisecond crystallography (SMX); viscous medium.

Figure 1

Schematic diagram of experimental geometry for serial crystallography using sample delivery medium. XFEL (X-ray free electron laser) or the synchrotron X-ray is focused using Kirkpatrick-Baez (KB) mirrors. The injection stream of the delivery medium containing crystals is extruded from the sample injector into the X-ray interaction point (red circle). The single panel detector without a center hole is the required beam stopper. Diffraction data is recorded by the detector.

Figure 2

Crystal growth in delivery medium for serial crystallography. Example of crystallization of membrane protein in lipidic cubic phase (LCP). (A) Monoolein (9.9 MAG) as a delivery medium and membrane protein solution is injected into each syringe. The ratio of monoolein and protein solution is 3:2. (B) Syringes containing the monoolein and protein solution are connected using a coupler. (C) Mixing of membrane protein and monoolein forms LCP. (D) Crystallization solution is added to the syringe containing the mixture of membrane protein in LCP. (E) Crystal growth in LCP. After removing the precipitant, the LCP containing the crystals is transferred into the sample injector and is used to perform the serial crystallography (SX) experiment. This figure was drawn based on Reference [42]. A horizontal arrow indicates movement of the plunger. In vacuum, an additional titration step using short MAG is required (see text).

Figure 3

Schematic representation of manual mixing of crystals and delivery medium. (A) The crystals and the delivery medium are mixed using a spatula under a glass slide. (B) The mixture is transferred to the dispenser tip. (C) The mixture is then moved to the end of the tip using centrifugation. (D) The mixture is transferred to the syringe or sample injector. This figure was drawn based on Reference [52].

Figure 4

Schematic representation of mechanical mixing of crystals and delivery medium. (A) Delivery medium and crystal suspension are injected into separate syringes. (B) The syringes containing the delivery medium and the crystal suspension are connected using a coupler. (C) The crystal and delivery medium are gently mixed. (D) The delivery medium containing the crystals is transferred into a sample injector to perform the SX experiment.

Figure 5

Chemical structure of monounsaturated monoacylglycerol lipids (MAGs). (A) Shorthand representation of N.T MAG, where N (neck) is the number of carbon atoms in the acyl chain between the ester and cis-olefin bonds and T (tail) is the number of carbon atoms between the cis-olefin bond and the end of the chain. (B) Chemical structure of short MAG lipid 9.7 MAG and 7.9 MAG, used in the SFX experiment in vacuum to avoid the Lc phase of 9.9 MAG.

Figure 6

Chemical structure of polysaccharide-based hydrogels. (A) Agarose, composed of β-(1-4)-(3,6)-anhydro-l-galactose (left) and α-(1-3)-d-galactose (right). (B) Hyaluronic acid (HA) composed of alternating residues of β-d-(1-3) glucuronic acid (left) and β-d-(1-4)-N-acetylglucosamine (right). (C) Structure of cellulose derivatives. In hydroxyethyl cellulose (HEC), R is H or hydroxy ethyl group (–CH₂CH₂–OH). In carboxymethyl cellulose sodium salt (NaCMC), R is H or carboxymethyl groups (–CH₂–COOH).

Figure 7

Chemical structure of polymer-based hydrogels. (A) Pluronic F-127. (B) Poly(ethylene oxide). (C) Polyacrylamide. Acrylamide and bis-acrylamide polymerize into polyacrylamide by adding the Tetramethylethylenediamine (TEMED) and ammonium sulfate.