Conversion of Vapor Diffusion Methods to Microbatch
When the conditions for growing crystals of a protein in vapor diffusion are known, crystals of comparable or better quality can usually be produced in microbatch by using the following guide-lines: (For microbatch, the concentrations of protein and other ingredients below refer to the concentration in the drop after protein and other ingredients have been mixed.)
In examples a - c, the concentration of protein quoted for vapor diffusion refers to the concentration of the stock solution before mixing with the reservoir solution. In all examples, the drop contained equal volumes of protein and reservoir solution.
a. Reverse Transcriptase
In the case of the full-length reverse transcriptase molecule, crystallized with salt in the form of hexagonal bipyramids, the concentration of the ammonium sulfate precipitant in the microbatch drop (1.3 M) was a little lower than the vapor diffusion reservoir (1.6 M). The other concentrations were the same. Note that a more concentrated protein stock solution was needed for microbatch.
b. Carboxypeptidase G2
In the case of carboxypeptidase G2, which was crystallized using PEG, the protein concentration in the drop was half the concentration of the stock used in vapor diffusion - therefore the same protein stock solution could be used. (The relatively high PEG concentration implies that some equilibration had occurred before crystallization took place in vapor diffusion.)
c. Glucose Isomerase from Bacillus coagulans
d. Erythrina Trypsin Inhibitor
* protein concentration shows actual concentration in drop after mixing, but before equilibration (i.e. stock was 20 mg/ml).Even in cases where proteins are crystallized by changing the pH using diffusion methods, it has generally been possible to grow good quality microbatch crystals. For example, Erythrina trypsin inhibitor was crystallized by dissolving the protein in sodium acetate buffer at pH 3.5, then equilibrating in sitting drops using a well solution containing buffer at pH 5.0. Crystals were grown in microbatch at pH 5.2, with a slightly higher protein concentration.
to Lesley Haire and Naomi Chayen who contributed these examples. Other
examples can be found in Naomi's paper: N.E. Chayen. Comparative
studies of protein crystallization by vapor diffusion and microbatch. Acta
Cryst. D54 (1998), pp 8 - 15.