Birefringence

Setting up an in meso crystallization trial is straightforward (Fig. 4 ▶). Typically, it involves combining two parts protein solution with three parts lipid at 20°C (Caffrey & Cherezov, 2009 ▶ ; Caffrey & Porter, 2010 ▶ ). The most commonly used lipid is the monoacylglycerol (MAG) monoolein. According to the monoolein–water temperature–composition phase diagram (Fig. 5 ▶; Qiu & Caffrey, 2000 ▶ ), and assuming there is no major influence on the phase behaviour of the protein-solution components, this mixing process should generate, by spontaneous self-assembly, the cubic mesophase at or close to full hydration. The original method for mixing lipid and protein solution involved multiple, cumbersome centrifugations in small glass tubes. Harvesting crystals required cutting the tubes and searching for small crystals through curved glass, which was not easy, very inefficient and required experience, time and patience.
The cubic phase is sticky and viscous in the manner of thick toothpaste (Fig. 6 ▶). As such, it is not easy to handle. In the course of earlier lipid-phase science work carried out in the Membrane Structural and Functional Biology (MS&FB) group, we had developed tools and procedures for manipulating such refractory materials. One of these, the coupled-syringe mixing device (Fig. 4 ▶; Cheng et al., 1998 ▶ ), was ideally suited to the task of combining microlitre volumes of monoolein with membrane-protein solution in a way that produces protein-laden mesophase for direct use in crystallization trials with minimal waste. The mixer consists of two, positive-displacement Hamilton micro-syringes connected by a narrow-bore coupler. Lipid is placed in one syringe and protein solution in the other. Mixing is achieved by repeatedly moving the contents of the two syringes back and forth through the coupler (Caffrey & Porter, 2010 ▶ ). The coupler is replaced by a needle for convenient dispensing of the homogenous mesophase into wells of custom-designed, glass sandwich crystallization plates (Cherezov & Caffrey, 2003 ▶ ; Cherezov et al., 2004 ▶ ). Precipitant solutions of varying compositions are placed over the mesophase and the wells are sealed with a cover glass. For initial screening, the plates are incubated at 20°C and monitored for crystal growth. The optical quality is the best it can be given that the mesophase is held between two glass plates and the mesophase itself is transparent (Fig. 7 ▶). This means that crystals of just a few micrometres in size can readily be seen by microscope whether the proteins are coloured or not. The use of cross-polarizers can enhance the visibility of small crystals, which usually appear birefringent on a dark background; the cubic phase itself is optically isotropic and non-birefringent. An added feature of the glass sandwich plates is that the double-sided tape used to create the wells provides almost hermetic sealing, ensuring minimal change in well composition during the course of trials that can last for months. Step-by-step instructions, complete with an open-access online video demonstration of the entire in meso crystallization process, have been published (Caffrey & Cherezov, 2009 ▶ ; Caffrey & Porter, 2010 ▶ ; Li, Boland, Aragão et al., 2012 ▶ ; Li, Boland, Walsh et al., 2012 ▶ ).

To establish the phase behavior and detect colloidal aggregates (>1 µm), VELM imaging of the samples was performed with an Olympus BX51 (Tokyo, Japan) microscope using bright-field illumination with differential interference contrast (DIC). The images were acquired with an Olympus DP71 digital video camera and the micrographs were processed with the Olympus CellA software provided by the manufacturer. The microscope was also used in polarized light mode to capture any birefringent structures (e.g., Maltese crosses, indicative of large multilamellar vesicles).