Precipitates from crystallization trials

During these trials, the chip also produced crystals of two targets that had not been seen by conventional screening. A previously unidentified crystal form of the bacterial 70S ribosome was obtained in three conditions of a sparse matrix of precipitants (Hampton Crystal Screen I), demonstrating that large protein-nudeic acid complexes may be crystallized in chip (C. Hansen, A. Vila-Sanjurjo and J. Cate, personal communication). Crystals of a previously uncrystallized mycobacterial RNase were also obtained from a single experimental condition on chip, whereas no crystals had been observed for this sample despite prior extensive... 

In practice, vapor diffusion crystallization trials are generally carried out using 24-well Linbro plates with each well sealed by a microscope cover slip of appropriate size (See Figure 2.2b). Each well of the plate contains 500 (rl of a precipitant solution. A 2- to 4- tl drop, containing equal parts protein solution (-10 mg/ml) and precipitant solution (taken from the well), is then placed on a siliconized microscope cover slip that is carefully inverted and placed over the well. A bead of high vacuum grease previously applied to the lip of each well provides the seal between the well and the cover slip. 

Although there exists a vast number of protein structures in the databases, there is as yet no rational procedure to crystallize a particular protein. The procedure is still mainly based on a trial and error approach. The crystallization process itself is one of which the protein is slowly and orderly precipitated from a solution. As a general rule, the purity of the protein is the most important factor to be dealt with before attempting to crystallize a protein. If possible, care should be taken not only to remove contaminant proteins, but also to remove any structurally heterologous populations in the purified protein sample. This may be achieved by discarding tail... 

A regularly formed crystal of reasonable size (typically >500 pm in each dimension) is required for X-ray diffraction. Samples of pure protein are screened against a matrix of buffers, additives, or precipitants for conditions under which they form crystals. This can require many thousands of trials and has benefited from increased automation over the past five years. Most large crystallographic laboratories now have robotics systems, and the most sophisticated also automate the visualization of the crystallization experiments, to monitor the appearance of crystalline material. Such developments [e.g., Ref. 1] are adding computer visualization and pattern recognition to the informatics requirements. 

The actual yield may be obtained from algebraic calculations or trial-and-error calculations when the heat effects in the process and any resultant evaporation are used to correct the initial assumptions on calculated yield. When calculations are made by hand, it is generally preferable to use the trial-and-error system, since it permits easy adjustments for relatively small deviations found in practice, such as the addition of wash water, or instrument and purge water additions. The following calculations are typical of an evaporative crystallizer precipitating a hydrated salt. II SI units are desired, kilograms = pounds X 0.454 K = (°F -I- 459.7)/I.8. 

Example of Application of the Growth Ramp. The experimental verification of the growth ramp equation, Eq. (6.56), was carried out by Karpinski (1996), for octahedral and cubic AgBr submicron crystals in a 6.0-1 batch reactor-precipitator. In the preliminary experiments, the number and size of the effective (stable) nuclei were determined. To this end, trial precipitations were performed with samples for sizing taken just prior to the growth stage and at the end of precipitation. The size was measured by a disc centrifuge-sizer, and the number of crystals was determined from the material balance. 

In the sitting drop method a small volume of protein solution is mixed with an equal volume of precipitant solution (which may be a concentrated solution of salts or other precipitants) and placed in a chamber surrounded by a larger volume of the precipitant solution. Water will evaporate from the protein droplet and pass through the vapour phase into the more concentrated precipitant solution. This gradually increases the concentrations of both the protein and the precipitants in the drop, until the precipitant concentration equals that of the surrounding reservoir. Under the right conditions (which are usually achieved by trial and error) this slow increase in concentration (typically over many days) allows growth of well-formed crystals. 

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