bkoep Staff Lv 1
It's time for an update on Foldit protein design! If you recall, our last update showed that several Foldit player-designed proteins appear folded and stable in solution. However, we'd like to have crystal structures of these proteins to show that they are indeed folding into their intended folds. The first step in getting a crystal structure is getting a protein crystal. Here we take a closer look at the protein crystallization process.
Above is a 96-well crystallization tray. We use a robot to rapidly set up crystallization experiments with 96 different conditions per tray. For this protein we set up four trays, to test a total of 384 crystallization conditions.
Each “well” in the 96-well tray is actually divided into four distinct regions. In the upper right, a square reservoir holds the mother liquor. The mother liquor is typically an aqueous buffer with some salt and a high concentration of precipitant. The reservoir is accompanied by three circular drop wells, each of which contains of drop of our protein sample mixed with the mother liquor. In this tray, the three drop wells are used to test different drop ratios, with protein and mother liquor combined in a ratio of 1:1, 2:1, or 1:2.
Each of the 96 wells is sealed off from the air and from neighboring wells. However, within a well, the three drops share an atmosphere with the reservoir, so that the drops can equilibrate with the reservoir by vapor diffusion. Over time, water evaporates from the drops and condenses in the reservoir. As the drop volume decreases, the protein concentration in the drop gradually increases. Eventually, the protein concentration reaches a critical point and the protein crystallizes.
In the drop above, we see several plate-like crystals radiating outward from a single origin. Most likely a small dust particle at the center served to “seed” the growth of all these crystals.
The crystals are not actually colored, per se, but exhibit birefringence—meaning that they refract light waves differently, depending on the orientation of the light waves with respect to the crystal lattice. When viewed through a microscope equipped with a light-polarizing filter, the birefringent crystals appear colored.
These crystals appear to be thin and plate-like, suggesting this particular crystal lattice extends readily in height and width, but less easily in depth. Sometimes, this is indicative of imperfections in the crystal packing, and can limit the quality of x-ray diffraction. To follow up, we’ll try to optimize the crystallization conditions by setting up a number of similar drops with slight alterations in the composition, in hopes that we get larger, more substantial crystals. However, there's a chance one of these crystals will diffract well enough to yield a crystal structure.
Once we have a nice, high-quality crystal that yields a good x-ray diffraction pattern, we can set about solving the crystal structure. A solved crystal structure will tell us definitively whether the protein folds up as the designer intended!
