Section 4.1.2.4. Vapour diffusion methods

Crystallization by vapour diffusion was introduced to structural biology for the preparation of tRNA crystals (Hampel et al., 1968). It is well suited for small volumes (as little as 2 µl or less) and has became the favoured method of most experimenters. It is practiced in a variety of forms and is the method of choice for robotics applications. In all of its versions, a drop containing the macromolecule to be crystallized together with buffer, precipitant and additives is equilibrated against a reservoir containing a solution of precipitant at a higher concentration than that in the drop (Fig. 4.1.2.1c). Equilibration proceeds by diffusion of the volatile species until the vapour pressure of the drop equals that of the reservoir. If equilibration occurs by water (or organic solvent) exchange from the drop to the reservoir (e.g. if the initial salt concentration in the reservoir is higher than in the drop), it leads to a volume decrease of the drop, so that the concentration of all constituents in the drop increase. The situation is the inverse if the initial concentration of the crystallizing agent in the reservoir is lower than that in the drop. In this case, water exchange occurs from the reservoir to the drop. Crystallization of several macromolecules has been achieved using this `reversed' procedure (Giegé et al., 1977; Richard et al., 1995; Jerusalmi & Steitz, 1997).

Hanging drops are frequently deployed in Linbro tissue-culture plates. These plates contain 24 wells with volumes of ∼2 ml and inner diameters of 16 mm. Each well is covered by a glass cover slip of 22 mm diameter. Drops are formed by mixing 2–10 µl aliquots of the macromolecule with aliquots of the precipitant and additional components as needed. A ratio of two between the concentration of the crystallizing agent in the reservoir and in the drop is most frequently used. This is achieved by mixing a droplet of protein at twice the desired final concentration with an equal volume of the reservoir at the proper concentration (to prevent drops from falling into the reservoir, their final volume should not exceed 25 µl). When no crystals or precipitate are observed in the drops, either sufficient supersaturation has not been reached, or, possibly, only the metastable region has been attained. In the latter case, changing the temperature by a few degrees may be sufficient to initiate nucleation. In the former case, the concentration of precipitant in the reservoir must be increased. A variant of the hanging-drop procedure is the HANGMAN method. It utilizes a clear, non-wetting adhesive tape that both supports the protein drops and seals the reservoirs (Luft et al., 1992).

Sitting drops can be installed in a variety of different devices. Arrangements consisting of Pyrex plates with a variable number of depressions (up to nine) installed in sealed boxes were used for tRNA crystallization (Dock et al., 1984). Drops of mother liquor are dispensed into the depressions and reservoir solutions with precipitant are poured into the bottom sections of the boxes. These systems are efficient for large drop arrays and can be used for both screening and optimizing crystallization conditions. Multichamber arrangements are suitable for the control of individual assays (Fig. 4.1.2.2). They often consist of polystyrene plates with 24 wells which can be individually sealed. Sitting drops can also be placed on microbridges (Harlos, 1992) or supported by plastic posts in the centres of the wells. Reservoir solutions are contained in the wells in which the microbridges or support posts are placed. Plates with 96 wells, sealed with clear sealing tape, are convenient for large-matrix screening. Most of these plates are commercially available and can often be used for a majority of different vapour diffusion crystallization methodologies (hanging, sitting or sandwich drops, the latter being maintained between two glass plates). A crystallization setup in which drops are deployed in glass tubes which are maintained vertically and epoxy-sealed on glass cover slips is known as the plug-drop design (Strickland et al., 1995). Plug-drop units are placed in the wells of Linbro plates surrounded by reservoir solution and the wells are then sealed as usual. With this geometry, crystals do not adhere to glass cover slips, as they may with sandwich drops.

Figure 4.1.2.2 | top | pdf | Two versions of boxes for vapour diffusion crystallization. On the left, a Linbro tissue-culture plate with 24 wells widely used for hanging-drop assays (it may also be used for sitting drops, dialysis and batch crystallization). On the right, a Cryschem multichamber plate, with a post in the centre of each well, for sitting drops.

Vapour phase equilibration can be achieved in capillaries (Luft & Cody, 1989) or even directly in X-ray capillaries, as described for ribosome crystallization (Yonath et al., 1982). This last method may even be essential for fragile crystals, where transferring from crystallization cells to X-ray capillaries can lead to mechanical damage. Vapour diffusion methods permit easy variations of physical parameters during crystallization, and many successes have been obtained by affecting supersaturation by temperature or pH changes. With ammonium sulfate as the precipitant, it has been shown that the ultimate pH in the drops of mother liquor is imposed by that of the reservoir (Mikol et al., 1989). Thus, varying the pH of the reservoir permits adjustment of that in the drops. Sitting drops are also well suited for carrying out epitaxic growth of macromolecule crystals on mineral matrices or other surfaces (McPherson & Schlichta, 1988; Kimble et al., 1998).

The kinetics of water evaporation (or of any other volatile species) determine the kinetics of supersaturation and, consequently, those of nucleation. Kinetics measured from hanging drops containing ammonium sulfate, polyethylene glycol (PEG) or 2-methyl-2,4-pentanediol (MPD) are influenced significantly by experimental conditions (Mikol, Rodeau & Giegé, 1990; Luft et al., 1996). The parameters that chiefly determine equilibration rates are temperature, initial drop volume (and initial surface-to-volume ratio of the drop and its dilution with respect to the reservoir), water pressure, the chemical nature of the crystallizing agent and the distance separating the hanging drop from the reservoir solution. Based on the distance dependence, a simple device allows one to vary the rate of water equilibration and thereby optimize crystal-growth conditions (Luft et al., 1996). Evaporation rates can also be monitored and controlled in a weight-sensitive device (Shu et al., 1998). Another method uses oil layered over the reservoir and functions because oil permits only very slow evaporation of the underlying aqueous solution (Chayen, 1997). The thickness of the oil layer, therefore, dictates evaporation rates and, consequently, crystallization rates. Likewise, evaporation kinetics are dependent on the type of oil (paraffin or silicone oils) that covers the reservoir solutions or crystallization drops in the microbatch arrangement (D'Arcy et al., 1996; Chayen, 1997).

The period for water equilibration to reach 90% completion can vary from ∼25 h to more than 25 d. Most rapid equilibration occurs with ammonium sulfate, it is slower with MPD and it is by far the slowest with PEG. An empirical model has been proposed which estimates the minimum duration of equilibration under standard experimental conditions (Mikol, Rodeau & Giegé, 1990). Equilibration that brings the macromolecules very slowly to a supersaturated state may explain the crystallization successes with PEG as the crystallizing agent (Table 4.1.2.2). This explanation is corroborated by experiments showing an increase in the terminal crystal size when equilibration rates are reduced (Chayen, 1997).