4 - PERSPECTIVE 2008 Nature Publishing Group...

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Protein crystallization: from purified protein to diffraction-quality crystal Naomi E Chayen & Emmanuel Saridakis Determining the structure of biological macromolecules by X-ray crystallography involves a series of steps: selection of the target molecule; cloning, expression, purification and crystallization; collection of diffraction data and determination of atomic positions. However, even when pure soluble protein is available, producing high-quality crystals remains a major bottleneck in structure determination. Here we present a guide for the non-expert to screen for appropriate crystallization conditions and optimize diffraction-quality crystal growth. We now live in an exciting era, where for the first time, biological systems are being understood at a molecular level. Because the functions of proteins are determined by their three-dimensional structures, protein crystal- lography has had a major role in shaping this under- standing (an illustration of the overall pipeline from gene to structure is given in Fig. 1 ). Often, the first bottleneck in the procedure is at the protein purification stage. The problem is how to obtain a concentrated pure solution (typically 5–15 mg/ml) of protein. Proteins have been made more amenable to crystallization by removal of flexible tails and interdo- main regions, as well as random or rational mutagenesis, especially for surface residues 1–3 . Even when pure soluble protein is available, produc- ing high-quality crystals is another bottleneck for struc- ture determination because crystallization is a complex, multiparametric process. The recent surge of structural genomics efforts, which aim to determine the structures of thousands of proteins, has encouraged the crystallog- raphy community to develop new methods for protein crystallization ( Box 1 ). Thus far, there are not any obvious correlations between crystallization conditions and protein struc- ture or family, nor are there any set rules or ‘magic bul- lets’ that will guarantee the production of good crystals. However, there are a variety of means to aid crystal- lization. Here we provide guidance to non-experts on protein crystallization—from screening for favorable crystallization conditions to optimizing crystal growth for production of diffracting crystals. We highlight state- of-the-art crystallization techniques that are simple and inexpensive and describe how to increase the chance of success by influencing the crystallization process. Setting up initial trials Finding crystallization conditions for a new protein is sometimes like searching for a needle in a haystack. The first step is to set up screening trials, exposing the protein to a variety of agents in order to find ‘hits’ or ‘leads’ that point to conditions that may be conducive to crystallization. Crystals, crystalline precipitate and phase separation are considered leads that are worth pursuing.
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