Converting VD to MB

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.)

Precipitant concentration is normally lower in the microbatch drop than in the vapor diffusion reservoir. Set up microbatch trials covering a range of concentrations from just below the concentration used in the reservoir, to half of this concentration.

Protein concentration is normally slightly lower than the stock solution used in vapor diffusion (i.e. the protein concentration before mixing it with well solution). Test a range from the concentration of the vapor diffusion protein stock solution to half this concentration.

In cases where crystallization takes place very rapidly, or where equilibration is slow (e.g. using high concentrations of polymers such as PEG), crystallization may take place in vapor diffusion before the diffusion equilibrium is complete. In such cases, the microbatch concentrations of protein and precipitant may be close to the concentrations in the vapor diffusion drop immediately after setting up (before equilibration takes place).

EXAMPLES

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

	Hanging drop		Microbatch
Protein	stock	10.0 mg/ml	droplet	10.0 mg/ml
HEPES buffer	reservoir	50.0 mM	droplet	50.0 mM
Ammonium sulfate	reservoir	1.6 M	droplet	1.3 M

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

	Hanging Drop		Microbatch
Protein	stock	6.0 mg/ml	droplet	3.0 mg/ml
Na cacodylate buffer, pH 6.3	reservoir	100.0 mM	droplet	100.0 mM
Zinc Acetate	reservoir	0.2 M	droplet	0.2 M
PEG 4K	reservoir	12.0%	droplet	10.0%

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

	Hanging Drop		Microbatch
Protein	stock	15.0 mg/ml	droplet	7.5 mg/ml
MES buffer, pH 6.0	reservoir	50.0 mM	droplet	50.0 mM
PEG 4K	reservoir	18.0%	droplet	9.0%

Another protein which was crystallized with PEG, glucose isomerase from Bacillus coagulans, crystallizes very rapidly (crystals are visible within 1 hour). In this case the microbatch protein concentration was half the concentration of the vapor diffusion protein stock, and the precipitant was at half the concentration of the reservoir. Apparently no significant equilibration had taken place in vapor diffusion before crystals appeared.

d. Erythrina Trypsin Inhibitor

	Sitting drop - drop	Sitting Drop - well	Microbatch
Protein	10.0 mg/ml*	-	12.0 mg/ml
Sodium acetate buffer	20.0mM, pH3.5	20.0 mM, pH5.0	50.0 mM, pH5.2
Sodium chloride	150.0 mM	50.0 mM	150.0 mM

* 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.

Many thanks 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.

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