Slow crystallization[ Rockefeller University]

The dried-droplet method will fail for samples containing significant concentrations of involatile solvents or high concentrations of salts [22]. The problem seems to be that as the droplet dries, the contaminants' concentrations become very high, interfering with matrix crystal growth and with protein incorporation. This concentration effect can be avoided by growing large, protein-doped matrix crystals under more controlled conditions. The technique described below has been shown to be effectiv e for protein solutions containing low protein concentrations (< 1 micromolar) and high concentrations of involatile solvents (e.g., 30% glycerol) or salts (4 M sodium chloride).

Large matrix crystals can be grown using the following procedure [22]. First, saturate the solvent mixture chosen for the mother liquor with the matrix at room temperature by placing 20 - 100 microliters of the solvent mixture (containing the protein and other solutes) in a 1.5 ml Eppendorf tube with an excess of matrix. Swirl the mixture with a vortex mixer for at least five minutes and then centrifuge the mixture to remove large crystals. Carefully remove the supernatant with a Pasteur pipette and sa ve it in a clean Eppendorf tube as the mother liquor. Heat the solution to approximately 40° C to remove any small matrix crystals remaining in suspension ¾ be sure to seal the Eppendorf tube! Remove the tube from the heater and allow it to come to room temperature before opening the tube. Place the opened tube into a holder on a vortex mixer's rotating stage and set the mixer to its lowest speed. Then allow the solvent to evaporate for several hours while swirling the tube at low speed. This procedu re results in a collection of protein-doped matrix crystals with long axes about 0.5 - 1 mm (for sinapic acid). Once the crystals have grown, remove the mother liquor from the Eppendorf tube with a Pasteur pipette. Wash the crystals by adding room tempe rature water and swirling the tube with a vortex mixer. Pellet the crystals by centrifugation and remove the solvent. Repeat the washing step several times. After the final removal of washing solvent, add small amount of water, slurry the crystals into the water and dump the contents onto a piece of filter paper or the sample stage.

There are many possible variations to this method. The saturated matrix solution may be prepared first and cleared of excess crystals prior to the addition of the protein to the solution, taking care not to dilute the matrix solution. Crystallization m ay also be carried out by lowering the temperature of the mother liquor by placing the open Eppendorf tube in a cold chamber or refridgerator.

Crystals can be mounted on a sample holder in a number of different ways. For small crystals (< 1 mm), transfer them to the sample holder while they are damp. The crystals usually adhere when as they dry. For larger crystals, it may be necessary to pl ace a very thin layer of polystyrene cement on the sample stage and adhere the crystals to the cement. Once they are mounted, the crystals can be examined using the same methods used for dried-droplet samples.