Cryopreservation of protein crystals has at least two advantages over room temperature methods. First, it greatly reduces radiation damage of the crystallized protein, especially when irradiated with higher intensity radiation sources. Second, it provides for relatively simple storage and transportation of crystals for remote data collection. Typical cryopreservants include glycerol, sugars (glucose and sucrose), and polyethylene glycols. Cryopreserved crystals are usually stored at liquid nitrogen temperatures (77K). Cryopreservants prevent the formation of ice in the cooled crystal and supporting mother liquor; instead of freezing when cooled, cryopreserved solutions vitrify into clear, supercooled glasses. Glycerol (30%) or glucose (25%) is usually sufficient to cryoprotect most crystallized proteins. Lower concentrations of cryoprotectants are necessary in the presence of high concentrations of salts or polyethylene glycol. Generally, if a drop of well solutions vitrifies to a clear glass in a sample loop, the cryoprotectant concentration is sufficient for ice formation suppression. The simplest method of cryoprotection is to simply transfer crystals directly from their mother liquor to a drop of artificial mother liquor with the added cryoprotectant. Soaking in the cryoprotectant drop for as little as 30 seconds is usually sufficient to prevent ice formation or crystal cracking. Longer soaks may be required if protein ligands are to be soaked in at the same time.
This method is especially appropriate for crystals that cannot tolerate direct transfer to cryoprotectant solution, or for crystals that are especially sensitive to concentration changes in the mother liquor driven by drop evaporation. In our laboratory this method is routinely used with success on otherwise very sensitive crystals. This particular method is adapted for hanging drop crystallization. Ligands can be soaked in at the same time as cryopreservation if included in the cryoprotectant solution at 125% of the final, desired concentration.
- Prepare a solution of artificial mother liquor + 30% w/v glucose (40% v/v glycerol or another cryoprotectant can be substituted)
- Remove a coverslip containing a drop with crystals to be cryoprotected and add 0.25 drop volume (DV) of cryoprotectant solutions (e.g. for a 4 uL drop add 1 uL of cryoprotectant solution). Replace coverslip on well and let stand for 5 minutes. Examine the crystals for cracking and/or dissolution.
- Repeat the previous step with the following additional cryoprotectant additions: 0.25 DV, 0.50 DV, 1.00 DV, 2.00 DV. After each addition replace the coverslip over the well and let stand for 5 minutes. Examine crystals for cracking and/or dissolution.
- After the last addition and 5 minute incubation, remove coverlip, fish out crystals with mounting loops and freeze directly in liquid nitrogen. The final glucose concentration will be 24%, sufficient to protect most crystallization solutions from ice formation upon freezing in liquid nitrogen.
protocol contributed by Roger Rowlett, Colgate University Department of Chemistry