Soaking, Mounting, and Freezing Protein Crystals
Roger S. Rowlett
Gordon & Dorothy Kline Professor, Emeritus
Colgate University Department of Chemistry
Gordon & Dorothy Kline Professor, Emeritus
Colgate University Department of Chemistry
Most X-ray crystallographic data collection is done at low temperature (typically 100 K) to minimize degradation of the crystal by free radicals generated by the X-ray beam. This is especially important when using intense synchrotron X-ray sources. In order to prevent crystals from cracking when frozen, it is necessary to treat protein crystals with a cryoprotectant prior to freezing. In the presence of a cryoprotectant, the protein and its thin layer of surrounding mother liquor will form an amorphous glass in which the crystal suffers minimal damage, and retains maximum X-ray diffraction properties.
Unless the optimum crystallization conditions already contain a sufficient quantity of cryoprotectant, it will be necessary to experimentally determine solution conditions suitable for safely freezing crystals. Typically, some quantity of cryoprotectant is added to a solution of artificial mother liquor, or a solution of artificial mother liquor containing the appropriate amount of cryoprotectant is made up from scratch. Some typical cryoprotectants and concentrations required to assure proper freezing protection in the worst-case scenarios is given in Table 1 below. In many cases a lower concentration of cryoprotectant that that listed in Table 1 is sufficient. (For example, crystallization solutions already containing high concentrations of PEG may require little or no additional cryoprotection.) The minimum amount of cryoprotectant required can be determined by taking up a small quantity of liquid in a crystal mounting loop and plunging it into liquid nitrogen. If drops reliably freeze clear, then the solution has sufficient cryoprotection for freezing protein crystals.
The choice of cryoprotectant will depend upon the crystallization solution composition. If protein crystallization conditions already contain a cryoprotectant, it is often ideal to simply increase the concentration to the appropriate value. This is especially convenient for PEG-containing solutions. However, PEGs have limited solubility in solutions that contain high concentrations of salt; in this case one of the other cryoprotectants in Table 1 is more likely to be suitable. Glycerol, glucose, or sucrose are very gentle to most proteins, have high solubility in a large variety of solution, and are often excellent choices.
Once a suitable cryoprotectant solution or solutions have been identified, the behavior of protein crystals in these solutions should be observed. This is often carried out at the same time as crystal mounting, as described below. You should observe that the crystal does not disintegrate, crack, or split during cryo-soaking. It is not necessary to soak crystals for extended periods to confer cryoprotection. All that is necessary is to replace the solution on the surface of the crystal with the cryoprotectant solution, a process that only takes a few seconds of soaking. For crystals that do not survive direct transfer into cryoprotectant solutions, try the "No-Fail" method of cryoprotection, described below.
Protein crystals are mounted for diffraction on tiny nylon loops 0.05–1.0 mm in diameter. The loops are mounted on hollow rods that are in turn mounted on magnetic caps that are conveniently stored under liquid nitrogen, and are easily placed on the goniometer head of the X-ray diffractometer. A photo of a loop and cap is shown below (Figure 1).
Figure 1. Mounting loops and cryovials
The following protocol is typical:
Notes
Occasionally, it is desirable to determine a protein structure in the presence of a bound small molecule. One method of preparing such protein-ligand complexes is to soak a crystal in artificial mother liquor containing an excess of ligand; this can be done at the same time as cryoprotection if desirable and practical. Typically, the concentration of ligand used should be 10-1000x the dissociation constant (Kd) if it is known. Soaking for 10-30 min should be sufficient to populate the protein in the crystal if the binding site is accessible in the crystal lattice. If protein molecules pack in the crystal in such a way as to obscure the ligand-binding site, or if crystals do not tolerate extended soaking without cracking or dissolving, then "no-fail" cryoprotection or co-crystallization with ligand should be attempted.
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. This method is typically gentle, but in our hands occasionally results in a slight but significant increase in crystal mosaicity. This increase in mosaicity can be reduced by shortening the soak time.
Notes