Crystallization trial

Every protein that is used in crystallographic studies is unique. The generalities that are offered in this section are just that, generalities. The amount of protein that is probably required when starting a crystallization experiment is about 10-20 mg/mL, and probably close to 1 mL for a reasonably complete survey of crystallization conditions. The actual concentration of protein needed for crystallization trials varies quite widely. Some reported crystallization conditions use less than 5 mg/mL of protein, while others use more than 20 mg/mL. The only way to determine the protein concentration required for a specific individual protein is through trial and error. 

Setting up a laboratory to run crystallization experiments will not require a huge investment of resources for the initial trials. This will allow to carry out the routine crystallization trials on a protein. If you are planning this to become a major portion of your research program, then a more significant investment will become necessary. The basic components required for crystallization are ... 

Incubator Crystallization trials are heavily dependent on the temperature and maintaining a constant temperature is critical to reproducible trials. In addition, crystallization trials are often carried out at different temperatures, the three most common temperatures being around room temperature (20-22°C), 12-15°C, and 4°C. Using a vibration-free incubator to maintain a constant temperature... 

Many proteins will not yield crystals in initial crystal trials. Unfortunately, crystallization is still a trial-and-error procedure, with no real way of predicting success or failure. If you fail to get crystals in your initial trials, it need not be the end of your structural studies (although it could be ). Many of the targets for neuroscientists are going to be membrane-bound or membrane-spanning proteins, and these are notoriously difficult to crystallize. Techniques are continuously being developed and refined to improve our ability to crystallize difficult protein examples. 

The first semi-high-throughput automated system to dispense crystallization trials of less than 1 jl1 was designed in 1990 to deliver batch trials imder oil (Chayen et ah, 1990). The method was named microbatch to define a microscale batch experiment. It was designed to obtain maximum information on the molecule to be crystallized while using minimal amounts of sample. In order to prevent the evaporation of such small volumes, the trials are dispensed and incubated under low density (0.87 g/cm ) paraffin oil (Fig. 3.2). The crystallization drops remain under the oil since the aqueous drops are denser than the paraffin oil. 

Protocol 3.10 Dispensing microbatch crystallization trials in gels... 

Chayen, N. E., Stewart, P. D. S. and Blow, D. M. (1992). Microbatch crystallization under oil - a new technique allowing many smaU-volume crystallization trials. J. Crystal Growth 122,176-180. 

Whatever the technique employed to purify the RNA, it is necessary to desalt and concentrate it prior to use in crystallization trials. Avery efficient way of achieving this is to use reverse-phase Sep-Pak columns that can be used on the bench (Waters Sep-Pak Cl8 Classic short-body). These are operated by gravity or using a syringe (Protocol 14.3). 

These problems are avoided by using an experimental design, where multiple factors are varied simultaneously between different crystallization trials. Each experiment n represents a combination Cn = xi,X2, , x0 of k factors. For each... 

A number of bioinformatic programs can calculate the distance between RNA secondary structures and perform clustering analysis to identify ensembles of related structures (Liu et al., 2008 Torarinsson et al., 2007). Partitioning the alignment in a set of ensembles thus reduces the total number of phylogenetic variants to be considered and single representative from each ensemble can be subjected to crystallization trials. 

An important feature of protein crystal growth experiments is the need to carry out crystallization trials with very small quantities of scarce and expensive materials. When experiments are carried out in such small volumes (typically, 5—100 ju.1), it becomes difficult to define and control solution properties. The situation becomes particularly complicated when vapor diffusion or other nonequilibrium approaches to crystal growth are used, as these produce different and changing conditions throughout the small volumes involved. 

The process of crystallization is still largely a process of trial and error, but in an attempt to formulate general rules, the cumulative experiences of X-ray crystallographers are being compiled into online databases such as the Marseille Protein Crystallization Database (14) and the Biological Macromolecule Crystallization Database (15). These and similar databases will provide useful starting points in crystallization trials and may greatly accelerate the process. 

Before crystallization trials, the protein was subjected to gel filtration on Superdex-75 (Pharmacia) in 50 mM sodium/potassium phosphate buffer, pH 7.4, containing 1 mM EDTA, 50 mM 2-mercaptoethanol, 150 mM sodium chloride, 5% glycerol and 5% 2-propanol, as described previously (12). The statine-based inhibitor, LP-149 (Ac-Nal-Val-Sta-Glu-Nal-NH2 e Nal is naphtylalanine and Sta is statine) (Fig. 1), was prepared at Lilly Research Laboratories (K. Hui, unpublished results). Crystallization was carried out at 4 °C using the hanging-drop vapor diffusion method as follows 2.5 //I of the FIV PR(D30N) at 7 mg/ml complexed with LP-149 (1 4 molar ratio) in 50 mM imidazole-HCl pH 7.0 containing ImM EDTA and 1 mM dithiothreitol were mixed with an equal volume of 2 M ammonium sulfate, 0.1 M sodium acetate, pH 4.6 (Hampton Crystal Screen, solution 47). Crystals appeared within a few days and reached the size of 0.2 x 0.2 X 0.4 mm in one week. 

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