A simple technique to reduce evaporation of crystallization droplets by using plate lids with apertures for adding liquids

Abstract

A method is described for using plate lids to reduce evaporation in low-volume vapor-diffusion crystallization experiments. The plate lids contain apertures through which the protein and precipitants were added to different crystallization microplates (the reservoir was filled before fitting the lids). Plate lids were designed for each of these commonly used crystallization microplates. This system minimizes the dehydration of crystallization droplets containing just a few nanolitres of protein and precipitant, and results in more reproducible diffraction from the crystals. For each lid design, changes in the weight of the plates were used to deduce the rate of evaporation under different conditions of temperature, air movement, droplet size and precipitant. For comparison, the state of dehydration was also visually assessed throughout the experiment. Finally, X-ray diffraction methods were used to compare the diffraction of protein crystals that were conventionally prepared against those that were prepared on plates with plate lids. The measurements revealed that the plate lids reduced the rate of evaporation by 63-82%. Crystals grown in 5 nl drops that were set up with plate lids diffracted to higher resolution than similar crystals from drops that were set up without plate lids. The results demonstrate that plate lids can be instrumental for improving few-nanolitre crystallizations.

Keywords: acoustic droplet ejection; crystallization; dehydration; high-throughput screening; in situ X-ray data collection; vapor diffusion.

Figure 1

Plate lids. Four plate lids that were designed to fit four popular crystallization plates were tested (similar designs that were not tested are available for many other crystallization plates). The plate lids were constructed out of 1 mm thick acrylonitrile butadiene styrene (Amtek P430) using a three-dimensional printer (Stratasys Dimension Elite).

Figure 2

Approach for evaporation control and strategy for calculating the evaporation rate. The plate lids that we designed channel the reservoir vapor pressure over the crystallization shelf in order to shield the crystallization droplet from dehydration by room air (a). To deconvolute the evaporation rate of the crystallization droplet from the reservoir, we used an analytical balance to periodically weigh crystallization plates containing either water or three types of mother liquor in two setups as shown in (a) and (b). We postulated that the small droplet on the crystallization shelf negligibly shielded the larger volume in the reservoir, so that the difference between the two measured evaporation rates was the evaporation rate for the droplet by itself (Evap_shelf ^deduced = Evap_A ^Obs − Evap_B ^Obs). We also directly measured the evaporation rate for uncovered plates Evap_C ^Obs as shown in (c).

Figure 3

Method for calculating the initial evaporation rate. We periodically measured the evaporation for droplets on covered and uncovered plates. The evaporation rate was calculated for droplets on the crystallization shelf of the uncovered plates and compared with the deduced evaporation rates for droplets on the crystallization shelf of plates covered with lids (using the technique described in §2.1). These data were plotted as a function of the 6½ h measurement time for each of our tested designs. The initial rate of evaporation was obtained from the y intercept of the least-squares fit between a fifth-order polynomial and the evaporation-rate data. The data below were obtained from a 7.5 µl droplet on the crystallization shelf of a CrystalQuick X plate (this data point is boxed in Fig. 4 ▶).

Figure 4

Plate lids reduce the rate of evaporation. Rate values are the initial rates of evaporation determined from the y intercept of a fifth-order polynomial that was least-squares fitted to each data set as described in Fig. 3 ▶ (the data used in Fig. 3 ▶ to illustrate the method for determining the y intercept are boxed).

Figure 5

The effect of air currents on evaporation. Air currents (taken from room air) greatly increase the evaporation rate for uncovered plates. The evaporation rates for all tested plates that were not fitted with lids (shown in orange) were much larger than the evaporation rates for the same plates with lids (shown in green).

Figure 6

Smaller apertures reduce evaporation rate. We compared the measured evaporation rate for uncovered plates (orange) with the evaporation rate for plates that were covered with plate lids that had square apertures with 0.88 mm sides (left), 0.66 mm sides (middle) and 0.44 mm sides (right). The smaller apertures further reduced the deduced rates of evaporation (green) at the cost of requiring greater precision from the plate-preparation robot.

Figure 7

Plate lids result in better and more reproducible crystals. The figure shows crystals that were grown using 2.5 nl precipitant solution and 2.5 nl lysozyme (a) and trypsin (b) solution in CrystalQuick plates (approximately 2½ min for the Echo 550 to prepare each plate). Each type of protein was prepared on a plate that had a lid and on a plate that did not have a plate lid. On average, using a plate lid resulted in fewer but larger crystals of lysozyme and trypsin (left). The number of thaumatin crystals was approximately constant. X-ray data were obtained from one of the lysozyme crystals in each well in the middle row of the plate (row D). The data from crystals prepared using lids were merged and compared with the data from crystals prepared without lids. We also used the technique described in §2.1 to measure the evaporation rate for each of the three types of mother liquor with no plate lid, and compared this with the deduced evaporation rate with a plate lid (right). Using lids during the 2½ min required for the Echo 550 to set up the 5 nl crystallization droplets allowed better, more reproducible and better diffracting crystals to be obtained.