Crystal Growth

Several methods are available for growing micro- or nanocrystals and preparing for a serial crystallography experiment.

Vapor diffusion crystallization

Schematic illustration of a vapor diffusion experiment, using a hanging or sitting drop configuration.
Schematic illustration of a vapor diffusion experiment, using a hanging or sitting drop configuration.

Vapor diffusion crystallization is a popular method for obtaining crystals of biological macromolecules. The protein solution is mixed with reagents that cause the proteins the crystallize, called the precipitant solution. This mixture is then placed—either as a hanging drop on a coverslip or as a sitting drop on a pedestal—over a reservoir that contains the undiluted precipitant solution. The hanging/sitting drop equilibrates with the reservoir solution through water vapor leaving the drop. This slowly increases the protein and reagent concentration in the drop, until supersaturation is reached, and crystals start to form.

Using a vapor diffusion setup, proteins can also be crystallized using supersaturation-controlled crystallization. By allowing the drop to dehydrate anywhere from 30 seconds to 30 minutes before sealing off the drop and crystallization well to increase the precipitant concentration in the drop. Allowing the drop to dehydrate for a longer time tends to cause more microcrystals.

Although vapor diffusion methods are often the first choice for screening crystallization conditions and growing large macromolecular crystals, they are less well suited for preparing serial crystallography samples. The hanging/sitting drop is usually only a few microliters in volume and several drops need to be combined to provide a sufficient volume with enough crystals for serial crystallography beamtime.

Simple batch crystallization

As an alternative to vapor diffusion crystallization, the batch crystallization method can be used. In the simplest batch crystallization experiment, the protein solution is directly mixed with the precipitant solution in their final concentration. In case the protein is crystallized in a very small volume, the drop can be covered with oil to prevent water evaporation.

The batch crystallization method can be used to crystallize large volumes of protein, making the method very suitable to prepare samples for serial crystallography experiments. To crystallize a large volume, the protein solution can simply be mixed with the precipitant solution and stored in a plastic microcentrifuge tube.

Rapid-mixing batch crystallization

For the rapid-mixing batch crystallization method a protein is required that can achieve high concentrations, typically >100 mg/ml. The precipitant solution is usually also concentrated as much as possible. The protein is added to a small glass vial with a stir bar at the bottom. The precipitant is then added in drops to the vial while stirring to ensure rapid equilibration. Typically, ratios of 1:3, 1:5, and 1:10 are tried to see which causes the most concentrated shower of microcrystals.

This technique is usually used when crystallization conditions are already well-known and have successfully been used to crystallize for example lysozyme and photoactive yellow protein.

Free interface diffusion crystallization

The free interface diffusion (FID) crystallization method is a batch crystallization method based on liquid-liquid diffusion. The protein solution is placed in a microcentrifuge tube and the precipitant solution is added in drops. The interface layer that forms between the two solutions is the nucleation point. The FID method works best if the protein solution is denser than the precipitant solution.

Crystallization by concentration

A simple method for exploring the phase space for crystallization concentrates the protein using a centrifugal filter. Starting with a low concentration protein solution, the protein is concentrated until approximately one-fifth of the volume. By diluting the concentrated protein solution back to the original volume with a precipitant solution and centrifuging again with a low centrifugal force (1000 rcf), the protein can be made to precipitate. If no precipitation is observed, more protein can be added to increase the concentration. This technique works best with a precipitant that is not retained by the centrifugal filter.

Lipidic cubic phase crystallization

The above crystallization methods are primarily suited for crystallizing soluble proteins and may be less successful when trying to crystallize membrane-bound proteins. Although membrane proteins can be solubilized in a detergent micelle and crystallized in a vapor diffusion experiment, many crystallization conditions need to be screened. The resulting crystals are often of lower quality than soluble protein crystals, limiting the resolution measured at the X-ray light source.

Instead of crystallizing the solubilized membrane protein, it is possible to stabilize the protein in a lipidic cubic phase (LCP), which spontaneously forms when lipids are mixed with water (or a protein solution) under controlled conditions. The LCP mimics the native membrane, thereby stabilizing the protein and increasing crystallization success. As a result, it has been possible to crystallize many challenging membrane proteins such as human G-protein coupled receptors (GPCRs) and obtaining high-resolution structural information on this important class of proteins.

More detailed information on LCP crystallization and protocols for crystallizing membrane proteins can be found on the webpage of the Cherezov lab.