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X-ray crystallography is the most successful methods for determining the structure of macromolecules. However, obtaining crystals of macromolecules is a very challenging task. The crystallization process can be considered as a two-stage experiment, where the first stage determines chemical and physical conditions under which the sample tends to crystallize, while the second stage refines the parameters to produce crystals suitable for analysis by X-ray diffraction. The quality and ease of X-ray structure determination is directly correlated with the size and the perfection of the crystalline samples. Initial crystals are frequently microcrystals or clusters which often have unfavorable morphologies or sizes, yielding poor diffraction intensities. It is therefore generally necessary to improve upon these initial conditions in order to obtain better crystals of sufficient quality for X-ray data collection.
Figure 1. Protein crystals of different quality and size obtained from an initial screen.
Creative Biostructure has extensive experiences in refining crystallization conditions after initial screens. We use the grid screen or incomplete factorial approaches in the optimization process, in which sequential, incremental changes are made in the chemical parameters that influence crystallization, such as pH, ionic strength and reagent concentration, as well as physical parameters such as temperature, sample volume and overall methodology. Optimization can also entail the application of novel procedures and approaches that may enhance nucleation or crystal development. Our fully automated robotics allow us to complete combinatorial mixing from a wide range of stock solutions, followed by many possibilities for dispensing nano-volume screening experiments in the 96 well format.
Table 1. Optimization variables and methods
|Variables||Precipitant type and concentration, pH & buffer, protein concentration, additives, drop volume & drop ratio, temperature and pressure|
|Methods||Microseeding, crystallization method, proteolysis, modification of the protein, kinetic modulation, combination screening, data & literature review|
Our crystallographers at Creative Biostructure perform optimization screening by careful manipulation and evaluation of biochemical, chemical, and physical variables, to produce crystals of sufficient size and quality for diffraction. We also provide guidance for our customers on how optimization might best be applied to crystal growth problems, and what parameters and factors might worth exploring to accelerate and achieve success.
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McPherson A, Cudney B. (2014) “Optimization of crystallization conditions for biological macromolecules. Acta Crystallogr F Struct Biol Commun 70(Pt 11):1445-1467.