Rational and Irrational Approaches to Convince a Protein to Crystallize

The importance of structural biology has been highlighted in the past few years not only as part of drug discovery programs in the pharmaceutical industry but also by structural genomics programs. Mutations of human proteins have been long recognized as the source of severe diseases and a structural knowledge of the consequences of a mutation might open up new approaches of drugs and cure. Although the function of a protein can be studied by several biochemical and/or biophysical techniques, a detailed molecular understanding of the protein of interest can only be obtained by combining functional data with the knowledge of the three-dimensional structure. In principle three techniques exist to determine a protein structure, namely X-ray crystallography, nuclear magnetic resonance spectroscopy (NMR) and electron microscopy (EM). According to the protein data bank (pdb; http://www.rcsb.org) that provides a general and open-access platform for structures of biomolecules, X-ray crystallography contributes more than 90% of all structures in the pdb, a clear emphasis of the importance of this technique. To perform X-ray crystallography it is essential to have large amounts of pure and homogenous protein to perform an even today still “trail and error”-based screening matrix to obtain well diffracting protein crystals. Therefore, successful protein crystallization requires three major and crucial steps, all of them associate with specific problems and challenges that need to be overcome and solved. These steps are (I) protein expression, (II) protein purification and (III) the empirical search for crystallization conditions. As summarized in Figure 1, every single step needs to be optimized along the long and stoney road to obtain protein crystals suitable for structure determination of your “most-beloved” protein via X-ray crystallography. This chapter will focus on these three steps and suggests strategies how to perform and optimize each of these three steps on the road of protein structure determination.