Crystallization conditions for

Here are discussed initial crystallization conditions for screening by robots using microplates. Three types of crystallization robots are presented. They were chosen for their distinct features, which give a picture of the technologies currently available. Crystallization conditions refinement methods based on statistical scoring have been discussed elsewhere (Francois et ah, 2004). 

Table 15.1 Crystallization conditions for DNA-protein complexes using polyethylene glycol (PEG) as a precipitant... 

Protein crystals suitable for soaking. Preferable if known inhibitor(s) have already verified the suitability of the crystals for soaking. If co-crystallization with the fragments is to be used instead of soaking, sufficient protein to grid around a robust crystallization condition for all fragments of interest is required. 

If co-crystallization is indicated by properties of the protein or the fragment library, then prepare a solution of the protein with suitable concentration of fragment(s), (suggested 100 mM). Screen this solution around known crystallization conditions for the protein. If no crystals are observed a full screen using numerous conditions may be indicated. 

An example of this approach is illustrated in Fig. 3.4. The graph in the center is a two-dimensional slice of a four-dimensional surface over which [protein], ([protein] X [precipitant]), pH, and temperature were varied, in attempts to find optimal crystallization conditions for the enzyme tryptophanyl-tRNA synthetase. Note that this surface samples the rectangular region [protein] versus ([protein] X [precipitant]), mentioned earlier. The height of the surface is the score for the crystallization. Surrounding the graph are photos of typical crystals obtained in multiple trials of each set of conditions. None of the trial conditions were near the peak of the surface. The... 

Table 1 shows the optimal compositions of the initial reaction mixtures and crystallization conditions for obtaining highly crystalline single phases of the borate compounds. 

The important point is that the determination of the crystallization conditions for various polymorphic forms need not be a completely random process. The combination of keen thoughtful observation with consideration of all the available crystal structures and thermodynamic information can provide extremely useful guidelines, if not for success, then at the very least for further experiments. Crystallization is almost never a sure-fire procedure, especially when one is trying to selectively produce a particular polymorph, and one that has proven consistently or suddenly elusive. 

A variety of methods have been employed to co-crystallize biological molecules with small molecules. Discovery of crystallization conditions is still an often tedious task, so newer methods for screening crystallization conditions for proteins include the use of semi-automated robots. 

From the point of view of purification, occlusion presents a serious obstacle in rejecting the impurities and residual solvent. Since solvent and temperature can drastically affect crystallization behaviors, these variables can play critical roles. Therefore, systematic screening of solvent and identification of proper crystallization conditions for optimum rejection of impurities are desirable. In view of the rapid development of high-throughput screening devices for the measurement of solubility (see Section 2.1.6), it is expected that there will be signih-cant progress in this field in the near future as well. 

The complex interactions between supersaturation, nucleation, and growth are critical to all crystallization operations. The critical properties of organic compounds that affect these three characteristics include the nucleation rate, growth rate, and width of the metastable zone. These properties vary over large ranges because of the complexity and variety of molecular structures. Crystallization conditions for a particular compound are therefore species dependent and difficult to predict without experimentation. 

Crystallization conditions for the preliminary X-ray work were described by Yonetani and Theorell (98-98) for binary complexes with NADH, ADP-ribose and 1,10-phenanthroline as well as for the ternary complexes E-NAD -pyrazole, E-NADH isobutyramide, and E-ADP-ribose-1,10-phenanthroline. Crystals of both the apoenzyme and these complexes suitable for detailed X-ray diffraction studies were prepared using pure isozyme EE by precipitation with ethanol (99) or 2-methyl-... 

Metabolic pathways of microorganisms Portal to ProTherm (protein thermodynamics), ProNit (protein-nucleic acid interactions), biomolecule structures Crystal data and crystallization conditions for proteins, nucleic acids, and complexes Enzyme nomenclature and properties See B.5... 

Polymorphism can result from conformational differences in a chain molecule or different packings of molecules with the same conformation. Such differences can be induced by slight alterations in the crystallization conditions, for example, by varying the crystallization temperature. 

At higher cooling rates the crystallization conditions for formation of the f form are less favorable. On the surface of the phthalocyanine pigment only a nudei are formed. The growth of nuclei leads to formation of the high crystalline structure built only from a crystals. 

On the contrary, up to now incorporation has been well assessed for elements which do not satisfy the above rule (e.g., B, Ga, Ti, Fe, see Table 2). Probably, at low concentration of the metal (concerning most cases), the geometrical parameters become less important than other factors related to the crystallization conditions. For these reasons it is not easy to identify simple criteria for predicting the ability of a given element to be incorporated in the silica framework. 

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