International
Tables for
Crystallography
Volume F
Crystallography of biological macromolecules
Edited by E. Arnold, D. M. Himmel and M. G. Rossmann

International Tables for Crystallography (2012). Vol. F, ch. 4.4, pp. 142-143

Section 4.4.5. Crystallization

K. H. Choia*

aDepartment of Biochemistry and Molecular Biology, 6.614C Basic Science, The University of Texas Medical Branch,University Blvd, Galveston, TX 77555–0647, USA
Correspondence e-mail: kychoi@utmb.edu

4.4.5. Crystallization

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HT crystallization using 96-well crystallization plates greatly reduces the total amount of protein required for screening and thus structure determination. Robotic crystallization systems are capable of dispensing nanolitre droplets (<100 nl) and hence substantially increase the number of conditions that can be screened with a fixed amount of protein sample, as well as reducing the time required for setting up a series of crystallization trials. A thousand conditions with different crystallization parameters (e.g. pH, salts, temperature) can be screened with 100 µl of protein sample. At the time of writing, commercially available crystallization robots include the Honey Bee (Cartesian Micro-array), Phoenix (Rigaku), Mosquito (Molecular Dimensions Limited) and Oryx (Douglas Instrumentation). These crystallization robots can set up either vapour-diffusion, microbatch or hanging-drop methods in 96-well plates within 15 min. Crystallization robots can also be integrated into a larger system that includes storage and imaging of crystallization plates, and liquid-formulation robots.

Honey Bee and Phoenix robots contain a single (or several) non-contact channel that dispenses protein solution and a 96-channel dispenser head that dispenses the crystallization solutions (50 nl to 100 µl). The 96-channel head transfers crystallization solutions from a 96-well deep-well plate into the reservoir and crystallization drop in a 96-well crystallization plate (Fig. 4.4.3.1[link]e). A single channel transfers protein solution into each of the 96-well drops one by one without touching the precipitate drops. The plate is then sealed with a clear film by the user. The Mosquito liquid-handling robot can set up drops with a hanging-drop geometry, and is more popular with membrane protein crystallization (detergent solutions have a tendency to adhere to the side of the drop well in sitting-drop geometry). The Mosquito uses only disposable tips capable of dispensing 20 nl–1.2 µl volume; thus the user can control the location of drop deposition more precisely because the disposable tips can touch the drop. The use of disposable tips also prevents cross-contamination between samples, and washing steps between samples are eliminated.

The Fluidigm TOPAZ system utilizes a new technology, the crystallization screen chip, in which protein sample and reagent solutions are automatically loaded into diffusion chambers within the protein screen chip and the two solutions mixed by free interface diffusion, as opposed to vapour diffusion or microbatch techniques (Thorsen et al., 2002[link]). A very small amount of protein is required for a crystallization screen, i.e. as little as 1.0 µl protein solution for 96 trials. Crystals obtained from the protein chip are generally too small for X-ray data collection, and thus need to be scaled up to obtain diffraction-quality crystals.

Progress of crystallization trials from large numbers of 96-well plates can be monitored using an imaging robot to take pictures of individual crystallization drops. The resulting images can then be analysed either manually or using automatic crystal recognition systems at specified time intervals (Markley et al., 2009[link]). Remote viewing of recorded crystal pictures is also available over the web. Each recorded image is linked to crystallization conditions for evaluation and scoring of the crystallization conditions (Fig. 4.4.3.1[link]f). Minstrel (Rigaku), CrystalFarm (Bruker) or HomeBase (The Automation Partnership) systems offer integrated systems for plate storage and imaging.

Crystallization conditions that initially produced crystals should be optimized to improve crystal growth and quality. A liquid-handling robot can be used to make screens in 96-well deep-well plates. A liquid-formulation robot has been developed for protein crystallization to make grid screens of 96-well deep-well conditions (e.g. Alchemist from Rigaku). The crystallization conditions stored in an imaging robot are linked to the liquid-formulation software, and can be used to formulate 96-well screen conditions for optimization experiments. Other optimization methods such as crystallization in gels, control of nucleation using oil mixtures or microporous materials, and seeding experiments can also be employed in an HT fashion (Chayen, 2003[link]; Georgiev et al., 2006[link]; Sugahara et al., 2008[link]).

References

Chayen, N. E. (2003). Protein crystallization for genomics: throughput versus output. J. Struct. Funct. Genomics, 4, 115–120.
Georgiev, A., Vorobiev, S., Edstrom, W., Song, T., Laine, A., Hunt, J. & Allen, P. (2006). Automated streak-seeding with micromachined silicon tools. Acta Cryst. D62, 1039–1045.
Markley, J. L., Aceti, D. J., Bingman, C. A., Fox, B. G., Frederick, R. O., Makino, S., Nichols, K. W., Phillips, G. N., Primm, J. G., Sahu, S. C., Vojtik, F. C., Volkman, B. F., Wrobel, R. L. & Zolnai, Z. (2009). The Center for Eukaryotic Structural Genomics. J. Struct. Funct. Genomics, 10, 165–179.
Sugahara, M., Asada, Y., Shimizu, K., Yamamoto, H., Lokanath, N. K., Mizutani, H., Bagautdinov, B., Matsuura, Y., Taketa, M., Kageyama, Y., Ono, N., Morikawa, Y., Tanaka, Y., Shimada, H., Nakamoto, T., Yamamoto, M. & Kunishima, N. (2008). High-throughput crystallization-to-structure pipeline at Riken SPring-8 center. J. Struct. Funct. Genomics, 9, 21–28.
Thorsen, T., Maerkl, S. J. & Quake, S. R. (2002). Microfluidic large-scale integration. Science, 298, 580–584.








































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