A successful crystallization trial

TABLE OF CONTENTS 1) Getting the most from your amino acid sequence.  2) Concentrating your protein. 3) Hanging drop vapour diffusion method of crystallization explained. 4) A Practical Guide to Crystallization by Mark Knapp.

There are several important questions the crystallographer should ask about a protein before beginning a crystallization experiment.  What is the moleular weight of the protein?  What buffer will the protein be stable in?  Will the protein remain active if I boil it in concentrated HCl? In the early days of crystallography, these questions could be directed to the biochemist who had painstakingly characterized the protein in full detail for many years in advance of structural studies.  Nowadays, with the advent of structural genomics projects, the amino acid sequence may be the only information a crystallographer has to go on. In this section, we explore how many of these important questions can be answered on the internet given only the amino acid sequence. 

Crystallographers are THE experts in protein concentration. Few other experiments in biochemistry require as high concentrations of pure protein as crystallization experiments. Crystallization typically requires 10 mg/mL concentration for proteins in the 10-30 kDa range. Larger protein tend to require less concentration (2-5 mg/mL). Smaller proteins tend to require higher concentration (20-50 mg/mL). There is a kit available to help you test whether your protein is at a suitable concentration for initial crystallization trials. It contains several precipitating agents commonly used in crystallization. If upon addition of these agents to 3 uL of your protein you find precipitation forming, then it is likely that your protein concentration is appropriate for crystallization trials. To get the Crystool Pre-Screen kit (free?), send e-mail to Bernhard Rupp.

I typically concentrate protein using a Centricon concentrator from Amicon. The centricon is designed to use centrifugal force to push solvent out through pores in a membrane, while your protein retained inside by size exclusion. The centricon holds up to 2.5 mL and concentrates down to a minimum of about 100 uL in an SS34 rotor. Concentration requires about 2 hours at 5000 rpm.

ALWAYS check the concentration of the protein before you use a centricon. Good recovery from a centricon is not always assured in the beginning, so you want to be able to check that you get the same amount of protein out of the centricon that you put in. Good recovery depends on chosing the correct molecular weight cutoff (MWCO), and the proper buffer: There are different MWCOs (3kDa, 10kDa, 30kDa) for different size proteins. The larger the MWCO, the faster the concentration process. Be careful to chose a MWCO that is significantly smaller than your protein's molecular weight. Otherwise, the protein may get trapped in the membrane pores. It is also a good idea to include at least 10 mM NaCl to prevent the protein from sticking to the surface of the membrane. If the ionic strength is too low, the hydrophobic parts of the protein glom onto the membrane surface, blocking the pores and slowing down the concentration process. When this phenomenon occurs, you could also significantly reduce the yield of protein recovery. 

Achieving an adequate protein concentration while maintaining solubility can be a tricky problem for some proteins. You may find that your protein "oils out" of solution. (i.e. You find a clear jelly at the bottom of the centricon recovery tube.) Or perhaps it precipitates in a white powdery cake. These things happen because the protein would rather interact with another protein molecule than with solvent molecules. The solution to the "non-solution" usually lies in finding the correct buffer pH or chemical additive. Change the pH of the buffer further away from the pI of the protein in order to increase the charge on the protein and the favorability of its interaction with solvent. Also, the addition of small polar organic molecules such as glycerol, sucrose, methylpentanediol, or 1,6-hexanediol often dissolves protein oils instantly. If you know that your protein uses metal ions, or binds a ligand, the addition of these chemicals could also improve protein solubility. In very difficult cases, you could use a mild detergent such as beta-octyl glucoside. A convenient way to test different compounds for their solubilizing effect on the protein is to take a drop of protein oil or precipitate on a glass coverslip and add some chemical to it while watching the drop in the microscope. If the oil or precipitate dissolves, then you can add that chemical to your protein stock before concentration to maintain solubility. 

(3) Hanging drop vapour diffusion method of crystallization explained

The hanging drop vapor diffusion technique is the most popular method for the crys tallization of macromolecules. The principle of vapor diffusion is straightforward. A drop composed of a mixture of sample and reagent is placed in vapor equilibration with a liquid reservoir of reagent. Typically the drop contains a lower reagent concentration than the reservoir. To achieve equilibrium, water vapor leaves the drop and eventually ends up in the reservoir. As water leaves the drop, the sample undergoes an increase in relative supersaturation. Both the sample and reagent increase in concentration as water leaves the drop for the reservoir. Equilibration is reached when the reagent concentration in the drop is approximately the same as that in the reservoir. 

See also other selected readings from Hampton Research.

For a thorough, well written article about current approaches to macromolecular crystallization, see Current Approaches to macromolecular Crystallization by Alexander McPherson, Eur. J. Biochem. 189, 1-23 (1990). 
You can get a copy from Mike.