Crystallization screens and methods

From CCP4 wiki

Because it is not generally possible to rationally predict crystallization conditions, screens have been developed which sample a wide range of crystallization space in order to identify preliminary crystallization condititions.

Many such screens are commercially available; they fall into several categories:

1. Sparse-matrix screens, which contain conditions that have previously proved successful.

2. Grid screens, in which specific crystallization conditions are sampled in a systematic way.

3. Imcomplete factorial screens, in which individual components of crystallization conditions are varied in a systematic way.


The PACT screen[edit]

The PACT screen was developed by Janet Newman in the lab of Tassos Perrakis. This systematic screen, a pH-, anion- and cation-testing (PACT) screen, aims to decouple the components of each condition and to provide information about the protein, even in the absence of crystals, rather than cover a wide crystallization space[1]. The PACT screen is available from Molecular Dimensions[2] and Qiagen [3] under license, and by Jena Biosciences[4].

The exact formulations and all information for how to make the PACT screen are available as supplementary material of the original article by Newman et al[5] and are available under Open Access policy.


Qiagen(Nextal) vs Hampton screens[edit]

Nextal Classics Suite is almost exactly the same as the Hampton Screen I and II (except that it's 96 conditions, whereas two Hampton Screens amount to 98; also, Nextal's screen is reorganized to bring closer the similar conditions for easier observation). Nextal Classics II Suite is almost exact replica of Hampton's Index Screen (even numbering is identical). One exception is the proprietary (!) Tacsimate in Hampton's Index, which in NExtal's screen is replaced (somewhat inappropriately) by sodium citrate.


What is Tacsimate?[edit]

"Tacsimate contains 1.36 M Malonic acid, 0.25 M Ammonium citrate tribasic, 0.12 M Succinic acid, 0.3 M DL-Malic acid, 0.4 M Sodium acetate, 0.5 M Sodium formate, and 0.16 M Ammonium tartrate dibasic."

What is Tacsimate? or go to the source

EDIT: The current "What is Tacsimate" document, at http://hamptonresearch.com/documents/product/hr000175_what_is_tacsimate_new.pdf indicates a slightly different formula:

"Tacsimate contains 1.8305 M Malonic acid, 0.25 M Ammonium citrate tribasic, 0.12M Succinic acid, 0.3 M DL-Malic acid, 0.4 M Sodium acetate trihydrate, 0.5 M Sodium formate, and 0.16 M Ammonium tartrate dibasic. This mixture is titrated to the appropriate pH using sodium hydroxide and is available in pH 4, 5, 6, 7, 8, or 9 reagent formulations."

Searching for silver bullets: An alternative strategy for crystallizing macromolecules. Alexander McPherson and Bob Cudney. Journal of Structural Biology 156 (2006) 387–406.

Desiccation of an existing screen which shows no sign of crystallization or precipitation[edit]

This reports a discussion on CCP4BB started on April 7, 2008 under the topic "which concentrated salt has lowest vapour pressure?".

If, after setup of a crystallization experiment and waiting long enough, the droplets stay clear, the question arises whether one can modify this experiment to still learn something. Thus the idea is that instead of setting up a new experiment (and losing the protein of the old experiment), one simply modifies the reservoir of the old experiment in an attempt to make the protein crystallize (or precipitate), and with the goal of bracketing the range of precipitant concentration. In this manner, one can "leap-frog" over the original conditions. However, the relationship between the chemical composition of the protein drop and that of the reservoir may not be maintained. Hence, the true crystallization conditions may not be simply the chemical composition of the reservoir. This idea has been explored in a number of publications:

  • H. Schreuder et al. published a method to measure the dehydration effect of precipitants (J. Appl. Cryst. (1988). 21, 426-429.). This publication has tables of the chemical potential of water in solutions of MPD, NaCl, potassium tratrate and PEG 6K.
  • similar experiments were done with NaCl, AS and MgSO4 by Arakali et al (Acta D 51 772-779, 2005)
  • AmSO4, NaCl and PEGs were explored in J. Newman, [6] Expanding screening space through the use of alternative reservoirs in vapor-diffusion experiments. Acta Cryst. (2005). D61, 490-493
  • AmSO4 didn't work well; the ammonia changes the pH: Dunlop & Hazes, Acta Cryst D61, 1041-8, (2005)
  • Use of dried seaweed; horse hair; cellulose and hydroxyapatite: Thakur AS, Robin G, Guncar G, Saunders NF, Newman J, Martin JL, Kobe B. [7] Improved success of sparse matrix protein crystallization screening with heterogeneous nucleating agents. PLoS ONE. 2007 Oct 31;2(10):e1091.
  • As an additive to the reservoir in a vapor diffusion experiment, LiCl can reduce the vapor pressure of water in a very controlled fashion. Other salts can be used; see the "Constant Humidity" table in the CRC Handbook of Chemistry and Physics (page E-46 in the 60th ed.). Adding LiCl reservoirs to microbatch plates can extend their useful lifetime by an extra week or two. For example: ~0.5 M LiCl matches the reduction in vapor pressure of water of ~40% PEG4000 in chemical potential, and ~1.5-2 M LiCl matches ~60-70% ammonium sulfate (~2.4 - 3 M).

Further findings/ideas:

  • LiCl seems to work very well in the reservoir, whereas in the droplet Li salts tend to increase the solubility of peptides (Seebach et al., Helv. Chim. Acta 72 (1989) 857-867) - but on the other hand Li salts can also be used as cryoprotectants.
  • at least 50% of PEG 4K, 8K, 10K desiccate drops. At less concentration PEGs seem to be poor.
  • add 100% glycerol or ethylene glycol
  • a number of substances [like dryrite, calcium sulfate (the salt that is used to absorb the humidity in damp rooms), and the gel-like stuff that keeps diapers dry] were mentioned, but they seem less appropriate to the author, for different reasons.