Protein Crystallization: Hanging and Sitting Drops Methods

In the previous chapter I mentioned that for the protein to crystallize, we need to bring the solution into a supersaturated state. The arrangement on the left shows how the hanging drop method is used. Normally, the concentration of the precipitant in the reservoir is higher than in the drop. Due to the concentration gradient, the water from the drop will evaporate towards the reservoir, thus lowering the volume of the drop and increasing the protein concentration. At some moment, the concentration of the protein will reach supersaturation, and if all other parameters like pH, ionic strength, type of buffer, additives, etc. are appropriate, the protein may start to crystallize. However, if we don't know the exact conditions for crystallization, we need to test a large number of conditions in order to find the most optimal combination of all parameters. Nowadays, people purchase crystallization screens and simultaneously set up hanging or sitting drops with a hundreds of conditions.
24-wells plates (Figure below) is used for setting up hanging or sitting drops in laboratory conditions. For extensive screening a 96-well plate is used, with small (nano-liter) drops requiring very small amounts of protein. Drops are usually disposed by special robots. For example, using a liquid handling robot, one may need as little as 15 microL of protein sample to screen 96 different crystallization conditions. The process is very quick and may only take a couple of minutes. Initial protein concentrations in the drops, at the start of the crystallization experiment, is variable and depends on the solubility of the protein - as a rule of thumb, more soluble proteins require higher concentration. Something between 5 to 10 mg/ml seems to be normal for most proteins. It is also possible to assess the required initial concentration using a so called pre-crystallization test (PCT), for example from Hampton Research. However, it is easy to design own pre-crystallization screens.
Some examples of crystals grown in a hanging drop experiment are also shown below. Crystals may need to be taken out from the drops to be placed into an X-ray beam. To protect the crystals from radiation damage, caused by high intensity X-rays, they are usually frozen at a temperature of liquid nitrogen and X-ray data are collected at these temperatures (cryo-temperature). Crystallization plates may also be placed directly into an X-ray beam at a synchrotron. This way data can be collected directly from unfrozen crystals. However, many crystals are required for this to work since each crystals may survive only one shut from the synchrotron beam.

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Crystal images are from Terese Bergfors’ site

Here some of the factors, which may affect protein solubility (and protein crystallization) are listed:

1) pH
2) Ionic strength
3) Concentration of precipitant
4) Concentration of macromolecule
5) Temperature
6) Additives, effectors, and ligands
8) Organism source of macromolecules
9) Presence of substrates, coenzymes, inhibitors
10) Reducing or oxidizing environment
11) Metal ions
12) Rate of equilibration
Even gravity has been considered as one of the possible parameters crystallization. NASA has a program for protein crystallization in space!

The list above may be extended and many more factors affecting protein solubility may be added. Commercially available crystallization screens may include hundreds of different solutions with various combinations of pH, ionic strength, salts, precipitants, various additives, metal ions, etc.
Apart from the method of hanging and sitting drops, other crystallization methods exist, although they are not so widely used for identifying initial crystallization conditions.

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Many new tools and methods for protein crystallization have been developed during the last 20 years. However, the methods of vapor diffusion, either with hanging or sitting drops, remains to be the most popular method when crystallization setups are considered. It is illustrated on the figure above.

Image from crystallization methods for proteins