TECHNOTES
Dehydrating Crystals Using the MicroRT^TM System

Dehydration has been used as a tool for inducing structural changes in protein crystals since the earliest days of protein crystallography. Max Perutz, John Kendrew and Hugh Huxley used it in their studies of hemoglobin and myoglobin in the 1940’s and 1950’s. Francis Crick was supposed to work on dehydration when he jointed Perutz’s group, but suffered a famous distraction.

Dehydration remains a powerful (and under utilized) tool for improving or at least modifying the diffraction properties of protein crystals. Dehydration removes excess solvent, tightens packing of protein molecules, and reduces the size of solvent channels. As a result, it sometimes improves crystal order and diffraction resolution at room temperature, and can make successful flash cooling easier, especially for crystals with large initial solvent contents. Protein crystals usually undergo structural transformations when sufficiently dehydrated, yielding alternative crystal packings that may be difficult or impossible to achieve directly during crystal growth. Of course, dehydration often severely degrades diffraction properties, but original crystal order can usually be fully recovered just by rehydrating.

The MicroRT™ system is the basis for an easy, controlled and reliable way to dehydrate crystals. The method is based on the fact that saturated solutions of salts, when placed in an enclosed container at constant temperature, will maintain a fixed relative humidity that depends on the salt. By equilibrating a crystal via vapor diffusion with a series of salt solutions, the solvent content can be reduced in a controlled and reproducible way, without the osmotic shock and other problems that occur when crystals are directly soaked in high salt or high PEG solutions. Here’s the procedure:

Prepare a series of saturated salt solutions corresponding to the desired relative humidities to be tested. As-grown protein crystals typically have relative humidities of 95-98%, so a good set of humidities to test is 93%, 86%, 79%, 75%, and 68%.

Inject a plug of 10-20 ml of the first saturated salt solution down toward the sealed end of a MicroRT™ capillary, and draw the capillary over your crystal and onto the goniometer base, as described in the “Using MicroRT™” instructions. If your crystal is to be flash cooled, do your cryoprotectant soak before starting dehydration.

Take single frames periodically to characterize how the diffraction changes as the crystal dehydrates. The equilibration time depends on the crystal size (but not on the equilibrium r.h.); for 100-200 μm crystals, equilibration will take 1-2 hours.

After equilibration with the first solution, just pop off the capillary, pop on another one filled with a salt solution that gives a lower final r.h., and take more frames. Repeat until you’ve explored a desired range of humidities. Then pop on the capillary with the salt solution (or the original stabilizing solution) that gives the best diffraction, equilibrate, and flash cool. To save time, you can dehydrate several crystals in parallel.

You should have little liquid remaining around the crystal, so there’s no need for additional cryoprotection. Some salt crystals may form around your crystal, but the diffraction spots from these can be easily eliminated from your analysis.

Click here for a list of saturated salt solutions and their relative humidities at 25°C.