Screening for crystallization conditions
Suppose we have a new protein to crystallize and don't know the exact conditions for crystallization (like the type of buffer, pH, salt concentration, etc.). In that case, we need to screen for crystallization conditions. At the start, we need to test many combinations with different duffers, different salts and salt concentrations, the effect of ligands, and different precipitants. The precipitant is the main component responsible for the creation of the "concentration gradient" required in vapor diffusion and other types of crystallization methods. Many different kinds of precipitants and ionic liquids are used in protein crystallography; probably the most common are ammonium sulfate, polyethylene glycols of various lengths, and propanol. Below is a table listing some common factors that affect protein solubility.
I have also downloaded an example of a screen
offered by Hampton Research to show the large number of salts and precipitants used in this screen alone. And there are a lot of other screens! This should give an idea of how protein crystallography works - many ready-made screens
are tested before the right crystallization conditions are found! We can purchase the crystallization screens and set up hanging or sitting drops with hundreds of conditions.
Specialized liquid handling robotics is used to set up nano-liter sitting-drop crystallization screens. An example is the Mosquito
liquid handling robot that is used with 96-well plates. A liquid handling robot may need as little as 15 microL of protein sample to screen 96 different crystallization conditions. The process is speedy and takes a couple of minutes. However, the initial conditions found from these screens often need to be further optimized before crystals useful for X-ray data collection are produced.
The protein concentration to be used in screening depends on the solubility of the protein. As a rule of thumb, the higher the solubility of the proteins, the higher the concentration should be. Remember that we need to reach a supersaturation level! The range between 5 to 10 mg/ml is expected 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. X-ray data collection
Grown crystals must be removed from the drops to mount them into an X-ray beam. To protect the crystals from radiation damage caused by high-intensity X-rays at synchrotrons, crystals are frozen at a liquid nitrogen temperature, and X-ray data are collected at these temperatures (cryo-temperatures). Sometimes data from crystals at room temperature are collected directly using the crystallization plates placed into an X-ray beam at a synchrotron. However, many crystals are required for this to work since each crystal may survive only one single shot of the high-intensity synchrotron beam. The next page gives some details of the X-ray crystallography experiment