Following the dawn of recombinant technology brought about by the groundbreaking overexpression of synthetic genes coding for insulin and somatostatin in Escherichia coli (Goeddel et al., 1979 ; Itakura et al., 1977 ) and the subsequent discovery of the polymerase chain reaction (PCR; Mullis et al., 1986 ; Saiki et al., 1985 , 1988 ), macromolecular crystallography was freed of its longstanding dependence on purified native protein samples for crystallization. Heterologous expression made it possible to generate samples of proteins and com­plexes that are found in only small or trace amounts in living cells and to engineer large and unstable proteins so that isolated domains or modified forms can be made available for crystallization. At the same time, the effort required for protein purification was dramatically reduced by the use of fusion proteins and affinity tags (Brewer et al., 1991 ; Sassenfeld, 1990 ; Malhotra, 2009 ). As a consequence, the overwhelming majority of samples used today for crystallization are recombinantly derived proteins. However, even though material for crystallization is more easily available, the prep­aration of single well diffracting crystals of the target macromolecule is still a time-consuming challenge.

Proteins are inherently dynamic entities, a property that greatly hinders their crystallizability. Not surprisingly, it has been estimated that even for the stable and relatively small single-domain prokaryotic proteins fewer than one in four will yield X-ray-quality crystals when using a routine screening process (Canaves et al., 2004 ; Price et al., 2009 ). In order to rationally modify proteins to enhance their crystallizability, it is first necessary to understand the physical properties that make most proteins resistant to crystallization.

The solubility of a protein is the primary essential prerequisite for its crystallization. It should be noted that the expression ‘low solubility’ is often used indiscriminately to describe quite different phenomena, including a propensity to aggregate and precipitate upon overexpression owing to misfolding, amyloid formation and finally genuine low in vitro solubility, i.e. low protein concentration in equilibrium with the solid phase, of otherwise fully folded and stable proteins (Trevino et al., 2008 ). Here, the strategies and methods that specifically address the latter case are discussed, i.e. precipitation at low concentrations of properly folded proteins.

The N- and C-termini of proteins are often flexible and un­structured (Thornton & Sibanda, 1983 ), creating a potential entropic impediment to crystallization. Initially, the preferred way to circumvent this problem was to use limited proteolysis to trim off the ends, leaving the stable core of the target protein. This strategy remains useful, particularly in its in situ version, in which trace amounts of proteases are added directly to crystallization screens (Dong et al., 2007 ; Wernimont & Edwards, 2009 ). However, on the downside it introduces the possibility of heterogeneity in the sample owing to incomplete proteolysis. An alternative route is to first identify the smallest functional fragment of the target protein and to then design and overexpress an appropriately modified gene. A number of options are possible. The simplest is the direct prediction of intrinsically disordered regions from the amino-acid sequence alone (Obradovic et al., 2003 ; He et al., 2009 ). The functional core units can also be identified experimentally by mass spectrometry following limited proteolysis (Cohen et al., 1995 ). Alternatively, deuterium–hydrogen exchange coupled to mass spectrometry (DXMS) may be used to identify fast-exchanging amides that map to unstructured fragments (Hamuro et al., 2003 ; Pantazatos et al., 2004 ; Sharma et al., 2009 ).

Tags are routinely used in heterologous protein expression in order to enhance folding and solubility and to facilitate purification (Uhlen et al., 1992 ; Malhotra, 2009 ). They are either short oligopeptides, such as a hexahistidine, with unique affinity properties or well expressed and highly soluble proteins, such as GST (glutathione S-transferase), MBP (maltose-binding protein) or thioredoxin. The tags are inserted into the expression vectors downstream or upstream of the target protein and are often separated from it by a protease-sensitive linker sequence. They are cleaved proteolytically following expression and partial purification of the fusion protein and removed, leaving the isolated target ready for crystallization. However, in some cases the target protein may not be adequately soluble after cleavage or may resist crystallization. One of the possible solutions is to use the intact fusion protein in the crystallization screens in the hope that the carrier protein will both confer solubility on the construct and mediate crystal contacts. Not surprisingly, the canonical carrier proteins, all of which crystallize fairly easily on their own, constitute the obvious first choice. Using this strategy, the DNA-binding domain of DNA replication-related element-binding factor (DREF) was crystallized in fusion with Escherichia coli GST (Kuge et al., 1997 ) and the U2AF homology motif (UHM) domain of splicing factor Puf60 was crystallized as a fusion with thioredoxin (Corsini et al., 2008 ). A key problem limiting the utility of this technique is the inherent flexibility of a two-domain fusion protein, which is detrimental to its crystallizability. A possible solution to this problem is shortening the linker between the two proteins until a relatively rigid construct is identified (Smyth et al., 2003 ). This approach was successfully pioneered for maltose-binding protein (MBP), which was used as a fusion chaperone to crystallize the human T-cell leukemia virus type 1 gp21 ectodomain fragment (Center et al., 1998 ). The same strategy was employed in the crystallization of the ZP-N domain of ZP3 (Monne et al., 2008 ), the islet amyloid polypeptide (IAPP; Wiltzius et al., 2009 ) and the MATα1 homeodomain (Ke & Wolberger, 2003 ). Recently, a genetically modified version of MBP (see below) was used as an N-terminal fusion chaperone to crystallize the signal transduction regulator RACK1 from Arabidopsis thaliana (Ullah et al., 2008 ; Fig. 2 ). Thus, MBP remains the most successful fusion chaperone for protein crystallization, even though the absolute number of proteins crystallized in this way is still limited.

Noncovalent crystallization chaperones, i.e. engineered binding proteins that produce noncovalent complexes with target macromolecules, constitute an exciting alternative to fusion carrier proteins. Complexes with such chaperones often exhibit enhanced solubility and/or crystallizability in com­parison to the isolated targets. The Fab and Fv fragments of antibodies are most commonly used for this purpose (Kovari et al., 1995 ; Hunte & Michel, 2002 ; Prongay et al., 1990 ; Ostermeier et al., 1995 ; Jiang et al., 2003 ; Dutzler et al., 2003 ; Lee et al., 2005 ). In its canonical version, the technique requires animal immunization with subsequent purification of hybridoma-derived antibodies and their proteolytic digestion to obtain pure homogeneous Fab fragments (Karpusas et al., 2001 ; Kovari et al., 1995 ). Alternatively, the Fab fragment can be directly sequenced and a synthetic gene can be used for E. coli expression, although this is not trivial owing to the presence of disulfides and two separate polypeptide chains in a Fab molecule. To overcome this bottleneck, a more efficient method of recombinant production of antibody fragments using mammalian HEK 293T has recently been proposed (Nettleship et al., 2008 ). Another possibility is the use of so-called nanobodies, i.e. single-chain fragments derived from camelid antibodies (Koide, Tereshko et al., 2007 ; Lam et al., 2009 ; Korotkov et al., 2009 ). However, this strategy requires immunization of camels or llamas, which is not technically easy.

A number of proteins undergo post-translational modifications which can adversely affect crystallization. By far the most ubiquitous is N- and O-glycosylation, primarily of membrane-associated, secreted and lysosomal proteins. In a number of cases successful crystallization of glycoproteins purified from natural sources has been reported and carbohydrate groups have often been found to be ordered and occasionally sequestered between the protein molecules, thus even contributing in a positive way to crystallization (Mark et al., 2003 ; Aleshin et al., 1994 ). In general terms, however, the flexible and heterogeneous carbohydrate moieties, particularly the oligosaccharides linked by N-glycosylation, can account for a significant fraction of the surface area of the protein and can therefore be detrimental to crystallization. The preparation of recombinant proteins in E. coli eliminates these post-translational modifications and may sometimes solve the problem (Mohanty et al., 2009 ), but N-glycosylation is often required for appropriate folding and solubility, so this approach is not always possible. However, if a eukaryotic expression system is a necessity, the problem can often be resolved by mutating the asparagines within the relevant glycosylation motifs (Asn-X-Thr/Ser), e.g. to aspartates, as was performed in the case of the extracellular domain of the metabotropic glutamate receptor expressed in insect cells (Muto et al., 2009 ), or to glutamines, as was performed for the human testis angiotensin-converting enzyme (Gordon et al., 2003 ). Alternatively, glycosylation at these sites can be eliminated by mutation of the Thr/Ser residues in the glycosylation motif to alanine or other amino acids, as described for rat cathepsin B (Lee et al., 1990 ), or valine, as was the case with the Ebola virus glycoprotein (Lee et al., 2008 , 2009 ). Similarly, potentially glycosylated threonines or serines in O-glycosyl­ated glycoproteins can be mutated to other amino acids (Horan et al., 1998 ) to avoid or reduce glycosylation.

There is currently no clear consensus regarding a possible correlation between the thermostability of a protein and its propensity to form crystals. It is often assumed on the basis of somewhat anecdotal evidence that thermostable proteins are more readily crystallizable and therefore if a specific target protein is recalcitrant to crystallization then a homologue from a thermophilic organism should instead be used. In some cases, low thermostability may correlate with the presence of unstructured loops or termini and consequently the construct-optimization strategies, as described above, are likely to yield a more crystallizable variant with a concomitantly increased stability. For example, a study of MAPKAP kinase 2 showed that truncated variants with increased thermal stability also showed higher crystallization propensity (Malawski et al., 2006 ). However, it is uncertain whether the relationship is causal or serendipitous. A recent analysis of large-scale data from a structural genomics project showed that when partly or fully unfolded proteins and hyperstable proteins (with melting temperatures T _m of greater than 363 K) are excluded from comparisons, thermostability per se does not correlate with propensity for crystallization (Price et al., 2009 ). Consequently, it appears that in a general case of a well behaving protein attempts to increase thermostability by site-directed mutagenesis may not necessarily yield variants with enhanced crystallization properties, although when the prospective crystallization target is inherently unstable engineering more stable variants may be helpful. In fact, this strategy has been successfully used for membrane proteins, which are often unstable in detergent environments. The first structure of a recombinant G-protein-coupled receptor (GPCR), i.e. bovine rhodopsin in complex with 11-cis retinal, was obtained using a thermostable variant with an engineered disulfide bond (Standfuss et al., 2007 ). In the more recent case of the turkey adrenergic β2 receptor, 318 variants were screened and six mutations were identified that increased thermostability. A variant containing all six mutations had an apparent T _m that was 21 K higher than that of the native protein in dodecyl­maltoside (DDM), was more stable in short-chain detergents and was successfully crystallized (Warne et al., 2008 , 2009 ). The effort required for such vast screening is substantial, but it appears that within protein families (such as GPCRs) the pattern of mutations enhancing thermostability is preserved, thus making it possible to transfer the mutations from one family member to another (Serrano-Vega & Tate, 2009 ).

For well ordered intrinsically stable proteins which show none of the problems addressed above, the propensity of the molecules to associate together and form crystals mediated by weak but specific interactions is ultimately defined by the physical chemistry and topology of the molecular surface. As already pointed out, large flexible amino acids on the surface, such as Lys, Glu and Gln, constitute an impediment to intermolecular interactions and consequently to protein crystallization (Cieślik & Derewenda, 2009 ; Price et al., 2009 ). In fact, it has been suggested that these residues and the ‘entropy shield’ that they form play a role in protein evolution which, given the high average concentration of proteins in cells, disfavors protein–protein interactions unless they are bio­logically functional (Doye, 2004 ). Thus, an intuitively obvious way to generate crystallizable variants is to replace selected large and surface-exposed residues with smaller residues such as alanine. This crystal-engineering strategy based on the surface-entropy reduction (SER) concept was extensively tested using as a model system the globular domain of the human Rho-specific guanine nucleotide-dissociation inhibitor (RhoGDI), which is recalcitrant to crystallization in its wild-type form owing to a high content of Lys and Glu residues, which constitute more than 20% of the sequence (Longe­necker, Garrard et al., 2001 ; Mateja et al., 2002 ; Derewenda, 2004 ; Cooper et al., 2007 ). These experiments established that in order to be most effective the SER strategy requires simultaneous mutations of clusters of two to three solvent-exposed high-entropy amino acids, typically Lys, Glu or Gln, located in close sequence proximity. These amino acids are replaced with alanine, although threonine and tyrosine, which is known to make a positive contribution at protein–protein interfaces such as antibody–antigen complexes (Fellouse et al., 2006 ), can also be used (Cooper et al., 2007 ). Engineered low-­surface-entropy variants of RhoGDI produced new and unique crystal forms, many with superior diffraction quality when compared with the wild-type protein. Importantly, in the vast majority of these crystals the mutated surface patches mediated crystal contacts, suggesting that SER engineering directly drives crystallization in a rational fashion by creating suitable crystal-contact-forming interfaces. The general utility of the method was further established by the crystallization of several novel protein targets found to be recalcitrant to crystallization in their wild-type form (Longenecker, Lewis et al., 2001 ; Derewenda et al., 2004 ; Devedjiev et al., 2004 ; Janda et al., 2004 ).

In most cases, protein engineering is used as a tool of last resort to obtain variants for proteins for which no crystals can be grown using the wild-type form. However, it may sometimes be necessary to obtain a new, different crystal form even when the wild-type protein does crystallize. Such a need may arise, for example, in drug-design investigations, where high-resolution structures are particularly critical for evaluation of the interactions between lead compounds and the target protein and may not always be possible using wild-type crystals. A novel crystal form may also be necessary if the wild-type crystals contain the target protein in an orientation in which the active site is obscured by crystal contacts, making it impossible to soak in drug lead compounds and screen small-molecule libraries by high-throughput crystallography (Blundell & Patel, 2004 ).

Protein engineering has become a routine tool that is used to generate crystallizable macromolecules and their complexes. While some approaches may only apply to very specific targets, a number of strategies offer general applicability. Among these, gene-construct optimization or surface-entropy reduction are quickly gaining popularity as methods of choice. However, it should be stressed that none of the methods described here offer a guarantee that the target protein can be coerced to crystallize. To maximize the chances of success, one must frequently attack the problem on multiple fronts based on an understanding of the chemical and physical properties of a specific protein. This is particularly true of technically difficult targets such as membrane proteins. A classic example illustrating this principle is the study of the HIV gp120 envelope glycoprotein (Kwong et al., 1998 , 1999 ). The con­struct that was ultimately used in successful crystallization screens had deletions of 52 and 19 residues from the N- and C-­termini and two flexible loops replaced by Gly-Ala-Gly linkages; additionally, the protein was 90% deglycosylated compared with the wild type. Moreover, this engineered gp120 was only crystallized in the form of a ternary complex with the CD4 receptor and a Fab fragment from a neutralizing antibody. In the recent case of the ATP-gated P2X4 ion channel, a crystallizable variant was obtained after a series of N- and C-­terminal deletions were screened to identify the smallest functional unit and the introduction of three mutations (C51F/N78K/N187R) to eliminate both aggregation arising from oxidation and N-glycosylation (Kawate et al., 2009 ).