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X-tal protocols

Screening for Crystals

Protein Detergent Methods Euipment Screen Drops Reference

The screening for suitable conditions is the search for a global or local maximum of a 'crystallization' function. This multidimensional phase diagram has many dimensions (factors) such as pH, temperature, precipitant, low ionic strength, mono- and divalent ions, etc.
The incomplete-factorial method is a powerful tool to reduce the number of crystallization trials required to identify the influence of different variables in crystallization experiments (Carter, 1979). To search the phase diagram a small number of random assigned experiments are used, scattered uniformly throughout the phase diagram, providing an even sampling of its entire volume. The conditions producing insoluble mixtures of precipitants must be eliminated. The experiments are performed in individual dialysis cells. The result of the solubility analysis represents the combined effects of the different factors. The effect of each individual factor is evaluated using a quality scoring and multiple regression analysis. The results are used for a more restricted search to improve crystal growth.

A coarse matrix to screen a large number of parameters with a limited amount of protein. The screen is based on the observation that promising leads can be identified over a broad range of parameters. The conditions are selected based on The set of conditions are designed by varying the parameters: buffer, precipitant and additive.
X-tal fact1 is based on a sparse-matrix procedure (Jancarik and Kim, 1991), conditions from the crystallization data bank (Gilliland, 1988) and results from the program Cristal (Roussell et. al., 1990)

flow chart screenings

Protein crystallization is carried out in several steps:

  1. The initial screen, for the identification of crystallization/nucleation conditions or promising leads.

  2. The adjusted screen, each conditions from the initial screen is adjusted to approach supersaturation more slowly.

  3. The optimization screen, based on the results found in the initial and adjusted screens, to obtain large single crystals (>0.2 mm) suitable for X-ray diffraction (better than 3Å resolution).

  4. To further improve the crystals, try additives.

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Initial screen.

For the identification of initial crystallization conditions, a flexible sparse matrix screen is used. In the initial screen we examine the roles of pH, precipitant, additives, and temperature.

Remember that the pH of the well solution is determined by the buffer, but also depend on the precipitant, additive and temperature used!

Prepare the stock solutions, consisting of different buffers, precipitants and additives, in 50 ml Falcon tubes. Filter through a 0.22 Ám filter and store at room temperature.

Although the preparation of wells from stock solutions is more labour intensive than using ready-made solutions, it has the advantage that precipitant concentration, pH and additive can be manipulated independently.

Make the screen by hand or by pipetting robot using the pipetting protocols:

For the initial screens the drop size is 1 µl protein and 1 µl well solution.

To prevent the drops from drying out, not more than three drops should be made at one time.

Place one screen at 4°C and one at 20°C, if the protein is stable at both temperatures. Cool the 4°C plates before pipetting the drops (to prevent condensation on the glass).

The screen is checked immediately and then every day (in the first week) to follow the precipitation/crystallization of the protein in time.

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Adjusted screen.

All wells, where the initial screen does not indicate nucleation, are repeated with the following modifications. Repeat until the protein precipitates between 2 days and 2 weeks (I once found crystals using 25 mM precipitant).

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Optimisation screen.

Optimisation Step 1

The conditions that favoured nucleation in the initial or adjusted screens are now used in the optimisation. For each condition, a new tray is set up in which one parameter is varied and the others kept constant:

The range for precipitant 1 in the rows A and B is usually between half and full concentration used in the initial or adjusted screen. Comparing the results from row A with B will show whether precipitant 2 is essential, and the optimal concentration of precipitant 1.

The result from row C will show the pH dependence.

Comparing the identical conditions from the optimisation tray, with the result from the initial screen, will give an indication of the reproducibility of the experiment. If the results from the comparable drops are not identical, check the protein stability.

Optimization Step 2

To refine the parameters from the optimisation screen, use a broad grid screen with variation in pH (in steps of 0.5 pH units) against precipitant. Refine the parameters until the optimal crystallization conditions are found. Remember: it is not size or morphology but the diffraction quality of the crystals that is important.

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Additives.

The crystals can be further improved by adding substate analogues, inhibitors, ligands or other additives:

5 % Jeffamine
5 % Polypropyleneglycol P400
5 % Polyethyleneglycol 400
5 % ethyleneglycol
5 % 2-methyl-2,4-pentanediol
5 % Glycerol
5 % Dioxane
5 % dimethyl sulfoxide
5 % n-Octanol
100 mM (NH4)2SO4
100 mM CsCl
100 mM CoSO4
100 mM MnCl2
100 mM KCl
100 mM ZnSO4
100 mM LiCl2
100 mM MgCl2
100 mM Glucose
100 mM 1,6-Hexanediol
100 mM Dextran sulfate
100 mM 6-amino caproic acid
100 mM 1,6 hexane diamine
100 mM 1,8 diamino octane
100 mM Spermidine
100 mM Spermine
0.17 mM n-dodecyl-ß-D-maltoside
20 mM n-octyl-ß-D-glucopyranoside


Top Back © 16 Febr 2004 Johan Zeelen