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.


Introduction
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.

Protein expression (I)
To crystallize a protein, the first requirement is the expression of your protein in high amounts and most importantly on a regular basis.This implies that it is possible to obtain a freshly purified protein at least weekly.In general, it is possible to express a protein either homologously or heterologously (see Figure 1 -(I) expression).Especially for large proteins, (PDB:3RYA) (Berntsson, Doeven et al. 2009) and the multidrug binding transcriptional regulator LmrR (PDB:3F8B) (Madoori, Agustiandari et al. 2009).

Eukaryotic expression hosts
The great benefit of choosing a eukaryotic host for overexpression of a protein of interest are the availability of a posttranslational modification system as well as the frequently enhanced protein folding (Midgett and Madden 2007).Eukaryotic proteins tend to misfold or lack biological activity when expressed in prokaryotic expression systems such as E. coli (Cregg, Cereghino et al. 2000;Midgett and Madden 2007).To overexpress these proteins, different yeast strains, insect cells or even mammalian cell lines have been developed as expression hosts (Figure 1 -(I) expression, right side).Eukaryotic expression systems are often more expensive, provide low expression levels and are sometimes hard to handle, when compared to bacterial systems.However, the genetic and cellular contexts are more similar to the original protein-expressing organism (Midgett and Madden 2007).In the following sections, some of the commonly employed eukaryotic expression systems will be described.

Yeast expression systems -Saccharomyces cerevisiae and Pichia pastoris
The most widely used yeast strains to express protein are Saccharomyces cerevisiae and Pichia pastoris, which offer the major advantage of a posttranslational modification system for glycosylation, proteolytic processing as well as disulfide bond formation, which for some proteins are essential for the function and/or correct folding (Cregg, Cereghino et al. 2000;Midgett and Madden 2007).The handling of yeast expression systems is similar to prokaryotic systems with respect to the genetic background and cultivation.Similar to the bacterial vector systems, expression in yeast starts with a plasmid-based cloning part which can be performed in E. coli (Cregg 2007).Afterwards the expression cassette gets integrated into the genome by simple homologous recombination in the yeast.One major advantage in P. pastoris is the insertion of multiple copies of the protein DNA-sequence into genomic DNA, which increases expression yield.The biggest advantage of yeast as expression system is that well established protocols for fermentation are available.Optimal fermentation of P. pastoris can end up with more than 130 gram of cells per liter of culture.Even if expression levels in the cell are not that high the mass of cells easily compensates for this disadvantage (Wegner 1990;Cregg, Cereghino et al. 2000;Hunt 2005;Cregg 2007;Midgett and Madden 2007).Examples of crystal structures from proteins expressed in P. pastoris are a human monoamine oxidase B (PDB:3PO7) (Binda, Aldeco et al. 2010) and a protein involved in cell adhesion NCAM2 IG3-4 (PDB:2XY1) (Kulahin, Kristensen et al. 2011).

Insect cells
The expression system in insect cells is beside yeast a well-characterised alternative to express eukaryotic proteins (Midgett and Madden 2007).As insect cells are higher eukaryotic systems their posttranslational modification machinery can carry out more complex alterations than yeast strains.They also have a machinery for the folding of mammalian proteins.The most commonly used vector system for recombinant protein expression in insect cells is baculovirus, which can also be used for gene transfer and expression in mammalian cells (Smith, Summers et al. 1983;D., L.K. et al. 1992;Altmann, Staudacher et al. 1999).A few examples of proteins expressed in insect cells that resulted in crystal structures are the transferase Ack1 (PDB:3EQP) (Kopecky, Hao et al. 2008), a human hydrolase (PDB: 2PMS) (Senkovich, Cook et al. 2007) and myosin VI (PDB:2BKI) (Menetrey, Bahloul et al. 2005).

Mammalian cell lines
The expression of proteins in mammalian cell lines is the most expensive and complex alternative.Especially for human membrane proteins this expression system has been proven to express the most active protein (Tate, Haase et al. 2003;Lundstrom 2006;Lundstrom, Wagner et al. 2006;Eifler, Duckely et al. 2007).The resulting protein amount, however, obtained from mammalian cell lines is mostly only sufficient for functional studies.Using mammalian cells lines is the most challenging variant of protein overexpression and therefore only choosen if any of the other expression system described failed.Some examples of protein structures expressed in mammalian cell lines are the hydrolase PCSK9 (PDB:2QTW) (Hampton, Knuth et al. 2007) and the acetylcholine receptor AChBP (PDB:2BYQ) (Hansen, Sulzenbacher et al. 2006).Table 1 sums up advantages and disadvantages of the above mentioned overexpression systems used for protein crystallography.

Purification
After having expressed your protein of interest, the race for crystals is by no means finished.The next step on the long road to structure determination is to isolate the protein orphrasing it differently -to remove all other proteins present in the cell (Figure 1 -(II) purification).An elegant method to do so is the genetic attachment of an affinity tag on either site of the protein or in some cases on both sides (Waugh 2005).This affinity tag has the possibility to bind high affine to a immobilized ligand on a matrix, while all other proteins have a much more reduced binding affinity and therefore flow through the matrix (Figure 1 -(II) purification 1 st step).This allows a one-step purification, which in almost all cases is relatively harmless for the protein and likely does not interfere with folding and/or overall structure of the protein.There are a lot of affinity tags available as well as matrix materials (Terpe 2003).The well known and most often used affinity tag is the poly-histidine tag (Porath, Carlsson et al. 1975;Gaberc-Porekar and Menart 2001), which can vary in length as well as in position but the overall purification strategy is the same.From all the structures solved nowadays, almost 60 % of the proteins are purified via a histidine tag; mainly due to the great purification efficiency, which can be as large as 90% after a single purification step (Gaberc-Porekar and Menart 2001;Arnau, Lauritzen et al. 2006).Therefore, most commercially available expression systems and methods contain a his-tag encoded on the plasmid.Besides the his-tag, there are other tags avaible and used for protein purification, of which the Strep-, CBP-, GST-, MBP-tag are described below.

Choice of the right tag 3.1.1 Polyhistidine-tag (his-tag)
As mentioned above the polyhistidine-tag is the most common affinity tag and the required affinity resins and chemicals are relatively inexpensive.The purification step is a so-called immobilized metal ion affinity chromatography (IMAC) (Porath, Carlsson et al. 1975).Here, a matrix is able to bind bivalent metal ions.For example nitrilotriactetic acid (NTA), which is a chelator and binds metal ions like Ni 2+ , Zn 2+ , Co 2+ or Cu 2+ (Hochuli, Dobeli et al. 1987).These metal ions have a high affinity to the imidazole group of the amino acid histidine.A stretch of histindines in a row with for example an E. coli protein is very unusual.Thus, the genetical introduction of several, in most cases 6-10 histidines in a row selects for specific binding of this protein.As eluant very elegantly imidazole can be used, which competes with the histidine tag and elutes the protein of interest.When used in low concentrations, imidazole can also be used to remove unspecifically bound proteins, which bind with low affinity to the matrix (Hefti, Van Vugt-Van der Toorn et al. 2001).Normally, a protein with a 6-10 histidine tag should be bound to the matrix relative strongly and 100-250 mM imidazole in the buffer is required to elute the protein from the resin.In contrast, proteins with a low affinity to the matrix can already be eluted with 10-50 mM imidazole (the "impurities" of E. coli).Therefore, a linear imidazol gradient, for example, separates the protein of interest and impurities (Hochuli, Dobeli et al. 1987;Gaberc-Porekar and Menart 2001).Although the polyhistidine-tag is the most common and mostly an efficient variant, there are a few applications where the his-tag can cause problems.Metalloproteins can interact either directly with the his-tag or with the ions immobilized on the matrix.In comparison to some other affinity-tags, the specificity of the his-tag is not that high and in some cases this results in the co-purification of other proteins (Waugh 2005).

Strep-tag
In comparison to the his-tag, which binds to immobilized metal ions, the strep-tag II constists of a small octapeptide (WSHPQFEK), which binds to the protein streptavidin (Schmidt, Koepke et al. 1996).The commercial available matrix is a streptavidin variant and is called Strep-Tactin.This variant is able to bind the Strep-tag II octapeptide under mild buffer conditions and can be gently eluted with biotin derivates such as desthiobiotin (Schmidt, Koepke et al. 1996;Voss and Skerra 1997).Especially for metal-ion containing enzymes it is a promising alternative to the his-tag (Groß, Pisa et al. 2002).However, as chemicals are more expensive and the matrix has a lower binding capacity, compared to NTA resins, it is often not the first option choosen.Moreover, it cannot be used under denaturating conditions since Strep-Tactin denatures and will not bind the tag anymore (Terpe 2003;Waugh 2005).Examples of proteins crystallized after a Strep-tag purification are OpuBC (PDB:3R6U) (Pittelkow, Tschapek et al. 2011) and AfProX (PDB:3MAM) (Tschapek, Pittelkow et al. 2011) as well as the sodium dependent glycine betain transporter BetP from Corynebacterium glutamicum (PDB:2WIT) (Ressl, Terwisscha van Scheltinga et al. 2009).

CBP-tag
Another peptide tag, is the calmodulin binding peptide, first described in 1992 (Stofko-Hahn, Carr et al. 1992).This peptide is prolonged compared to the Strep-tag II, consisting of 26 amino acids and binds with nanomolar affinity to calmodulin in the presence of Ca 2+ (Blumenthal, Takio et al. 1985).It is derived from the C-terminus of the skeletal-muscle myosin light-chain kinase, which makes the system an excellent choice for proteins expressed using a prokaryotic expression system, since in prokaryotic systems nearly no protein interacts with calmodulin.This allows extensive washing to remove impurities and elution with EGTA, which complexes specifically Ca 2+ , and a protein recovery around 90 % can be achieved (Terpe 2003).A drawback of this tag however is that the CBP tag can only be fused to the C-terminus of the protein since it has been shown that CBP on the Nterminus negatively influences the translation and thereby the expression rate (Zheng, Simcox et al. 1997).

GST-tag
With respect to the length of the tags, the his-tag contains only a few amino acids, the Streptag II and the CBP-tag already contain 8 -26 amino acids, but it is possible to fuse whole proteins with 26 -40 kDa to a recombinant protein.Here, the high affinity binding of the protein to their substrate is used to purify the protein of interest (Smith and Johnson 1988).In the case of the glutathione S-transferase (GST, 26 kDa) the protein specifically binds to immobilized glutathione.To elute the fusion protein from the resin, non-denaturating buffer conditions employing reduced glutathione are used (Terpe 2003).The tag can help to protect the recombinant protein from degradation by cellular proteases.It is recommended to cleave off the GST-tag after purification with a specific protease like thrombin or TEV (Tobacco Etch Virus) protease (Terpe 2003).

MBP-tag
Another affnitiy tag, which can be fused to the protein of interest, is the maltose binding protein (MBP) from E. coli.This protein has a molecular weight of 40 kDa and has the ability to bind to a cross-linked amylose matrix.The binding affinity is in the micro molar range and the tag can be used in a pH range from 7.0 -8.0, however, denaturating buffer conditions are not possible (di Guan, Li et al. 1988).The elution of the recombinant protein is recommended with 10 mM maltose.A great opportunity of the MBP-tag is the increasing solubility effect of the recombinant protein in prokaryotic expression systems and even more pronounced in eukaryotic systems (Sachdev and Chirgwin 1999).Like the CBP-tag, a fusion at the N-terminal side might influence translation and expression rates (Sachdev and Chirgwin 1999).

Tag position and double tags
As described above, the position of the tag either at the N-or C-terminus has a considerable influence on translation and expression rate as well as on the biological function (Arnau, Lauritzen et al. 2006).If information regarding activity of the protein is already available especially about the location of interaction sites, this should be included in the protein design, meaning tag position etc.In general, the tag should be placed at the position of the protein, which is less important for interactions and/or expression.To minimize the influence of the tag on folding and/or activity in some cases it helps to create a linker region of a few amino acids between the tag and the protein (Gingras, Aebersold et al. 2005).A very efficient and sophisticated solution is, the addition of amino acids between tag and protein of interest, which functions not only as an accessibility increasing factor, but, also encodes for a recognition site for proteases like thrombin or TEV.Due to this arrangement the tag -protein interaction is minimized and the tag can be cleaved off if necessary (Arnau, Lauritzen et al. 2006).In some special cases a combination of two affinity tags results in enhanced solubility and more efficient purification.To enhance the purity of a protein, often a construct of two different short affinity tags like his-tag and Strep-tag or CBP-tag can be engineered (Rubio, Shen et al. 2005).Also a combination of two his-tag or two strep-tag kept apart by a linker region enhances the binding affinity extremely.This allows more stringent washing steps prior to elution of the protein (Fischer, Leech et al. 2011).

Size exclusion chromatography and ion exchange chromatography
Despite the usage of affinity tags a second purification step is sometimes required (Figure 1 -(II) purification).Which kind of purification procedure is required depends on the nature of impurities.If these impurities differ in molar mass compared to the protein of interest, a method based on size separation can be applied.Size exclusion chromatography (SEC) also separates different oligomeric species of the protein from each other, which otherwise would strongly inhibit crystallization and also allows analysis of stability and monodispersity of the protein (Regnier 1983a;Regnier 1983b).However, in many cases, SEC is not sufficient to remove all impurities.Then separation by overall charge of the protein might be an option.Depending on the isoelectric point of the protein either anion or cation exchange chromatography can be performed.The protein binds to a matrix under very low ionic strength and is eluted afterwards either by increasing the ionic strength or by pH variation.Similar results can be achieved by hydrophobic interaction chromatography.Here, proteins with different surface properties show differences in their binding strength and binding of the protein is done inversely as during ion exchange chromatography.High ionic strength favors protein binding to a hydrophobic matrix and elution takes place when reducing the ionic strength.Although there are many other possibilities to increase the purity of a protein, the above mentioned techniques are without any doubt the most widely used and general applicable methods.

How to get a homogenous protein solution
In some cases isolated proteins are stable and homogenous at high concentrations after the purification and can be directly used for crystallization experiments.Often, however, the protein does not behave ideal and precipitates at high concentrations or forms aggregates or inhomogenous, oligomeric species; all of them prohibit crystal growth.SEC is a very elegant method to visualize the stability and oligomeric state of a protein.If the stability or the homogeneity of a protein sample is critical, you need to adapt your purification protocol and search for an optimized procedure.Different approaches are summarized below, for example a buffer screen to enhance protein solubility, multi-angle light scattering experiments to determine the absolute mass and the oligomeric state of the protein sample or fluorescence-based experiments to investigate the stability of the protein of interest.

Purified proteins -An in vitro system
After a protein is expressed in a soluble form, the subsequent purification procedure changes the environment of the protein dramatically.The cytoplasm of the cells, where the overexpression takes place, is packed with macromolecules.In E. coli, for example, the concentrations of proteins, RNAs and DNAs are about 320 mg/mL, 120 mg/mL, and 18 mg/mL, respectively (Cayley, Lewis et al. 1991;Zimmerman and Trach 1991;Elowitz, Surette et al. 1999) resulting in an overall concentration of macromolecules of above 450 mg/mL.During cell lysis and the first purification step, likely an IMAC (see above), the protein is separated from almost all other cell components.This rigorous procedure is accompanied with a severe change of the environment into an in vitro system.As a result proteins often tend to aggregate, precipitate or form inhomogeneous oligomeric states that prevent the formation of crystals in further experiments.Therefore one of the biggest challenges in structural studies is the preparation of protein solutions with high concentrations (as a rule of thumb 10-20 mg/mL) in a homogenous state.To fulfill these requirements, the in vitro system needs to be optimized with respect to different parameters as highlighted in Figure 1 -(II) purification.If a sufficient protein sample cannot be obtained, different strategies are available to increase the important characteristics of the protein: purity and homogeneity.As mentioned above, the usage of different metal ions during IMAC, ion exchange, a second affinity chromatography etc. can be sufficient to enhance purity.This might also lead to an increased stability.However, if the stability and/or homogeneity of a protein is still a problem, screening for a new buffer composition is essential to succeed during crystallization trials.

Buffer composition
Many examples illustrate the importance of an adequate buffer composition for protein stability, homogeneity, conformation, and activity (Urh, York et al. 1995;Holm and Hansen 2001;Jancarik, Pufan et al. 2004;Collins, Stevens et al. 2005).Some buffers are very frequently used and recommended by manufactures (see for example Qiagen, Roche, New England BioLabs, Fermentas, etc.).All of them contain a buffer reagent that keeps the pH constant in a well-defined range.Well-known examples are phosphate, tris (hydroxymethyl) aminomethane (Tris), or HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) that buffer at the physiological relevant pH range of 6-9 (Durst and Staples 1972;Chagnon and Corbeil 1973;Tornquist, Paallysaho et al. 1995).In recent years, the development of other buffer systems has been quite successful (Taha 2005) (for a list of buffers and corresponding pH ranges, see for example: http://delloyd.50megs.com/moreinfo/buffers2.html).Next to the well-defined pH, the stability and homogeneity of proteins depend on many other parameters, for example ionic strength, the presence of ligands and/or co-factors, divalent ions, glycerol, etc.The appropriate buffer composition cannot be predicted so far and needs to be identified by trial-and-error approaches.

Protein purification -How to overcome problems
In this part we would like to present some pitfalls that might occur during protein purification and provide some ‚rationales' to overcome these problems.As usual, the crucial step of solving a problem is its identification.Here, we are trying to sensitize the reader to indications, which might point towards problems related to instability and/or inhomogeneity of the protein sample.Moreover, such problems cannot always be recognized without the adequate technique(s).Therefore, we are introducing techniques that are capable to visualize the state of proteins.

Visible protein precipitations during IMAC
A very obvious stability problem is the formation of precipitations in the elution fractions of a chromatography step (see Figure 2).In this example, the his-tagged protein was eluted with a linear imidazole gradient from 10 to 500 mM imidazole and eluted at about 250 mM imidazole.Protein precipitation occured immediately after elution (Figure 2A and B) and continued (Figure 2C) resulting in a low amount of soluble protein.This aggregation can be reduced by dilution with a IMAC buffer (typically lacking imidazole) immediatly after the elution.Thereby, dilution hinders the concentration-dependent aggregation.In many cases, this rational is not sufficient to prevent precipitation.After applying, for example, a buffer screen (see Figure 1 -(II) purification) the new defined buffer is used for the chromatography or the eluting protein is diluted into the new buffer (see Figure 2D).Other elution strategies of his-tagged proteins from an IMAC column are available.As described before, competing the poly-histidine from the IMAC column by imidazole is the most common elution strategy, however, for some proteins other strategies are superior, for example, replacing imidazole by histidine.Imidazole is only a mimic for histidine.If one uses histidine instead of imidazole aggregation can be avoided as concentration of the eluent can be reduced by a factor of ten.An example for a protein sensitive to imidazole concentration is shown in Figure 3B.Here a comparative SEC chromatogram is shown.After elution from the IMAC column with imidazole only a very small amount of the protein elutes at the volume corresponding to the size of a monomer or the dimer, respectively (Figure 3B, continuous line).Most of the protein passes the column very fast and elutes at the void volume indicating large radii meaning aggregated protein.Yields of dimeric (at about 150 mL) and monomeric (at about 180 mL) proteins are strongly increased after an elution with histidine (dashed line) compared to an elution with imidazole (continous line) and only the monomeric species could be crystallized (data not shown).The choice of the eluent in IMAC might therefore be an important step in a purification protocol.Another elution strategy of his-tagged proteins is a pH change from 8 to 4. In an acidic environment, histidines become positively charged and are therefore released from the column matrix.This strategy results in a sharp elution from the matrix and the protein is eluted highly concentrated.Although this strategy is recommended by the manufacturers (see GE Healthcare, Qiagen, etc.) the desired protein needs to retain activity at acidic pHs.The bivalent metal ions (Ni 2+ , Co 2+ , Zn 2+ ,…, see above) that complex the his-tag can be removed from the matrix by chelating reagents as ethylenediaminetetraacetic acid (EDTA) as another elution strategy (Muller, Arndt et al. 1998)  The elution fractions were immediately mixed in a 1 to 1 ratio with a buffer that enhances the protein stability (50 mM citrate, 50 mM LiCl, pH 6.00) evaluated during a solubility screen.

Invisible aggregations
Sometimes aggregation of proteins in solution can not be detected directly by eye.This inhomogeneity of protein samples can be visualized SEC, a method that separates proteins by their hydrodynamical radius (see above).Protein aggregates are eluting at the void volume, since they are clumbed together resulting in a big hydrodynamical radius (see Figure 3A and B).If invisible aggregation is detected the buffer composition needs to be adjusted.In one case we applied this technique to visualize the state of a protein after an IMAC, and the resulting elution profile is shown in Figure 3A (continous line).Comparable to the imidazole-induced precipitation described above, the protein aggregated and elutes within the void volume of the column (about 40 mL).Moreover, several other protein species elute from 55 to 80 mL indicating a highly inhomogeneous protein sample.The running buffer of the SEC was 50 mM Tris-HCl, pH 8.0 and 150 mM NaCl.Remarkably, a simple change to a new buffer (20 mM HEPES, 150 mM NaCl, pH 7.0) Resulted in a stable and homogenous protein sample (Figure 3A, dotted line), which was suitable for crystallization trials.Next to the rigorous change in the homogeneity of the protein, the biological activity of the protein could only be determined in the new buffer system.The influence of the buffer composition for the protein activity is a well-known phenomenon (Urh, York et al. 1995;Holm and Hansen 2001;Zaitseva, Jenewein et al. 2005) and in many cases the activity goes hand in hand with an optimal buffer for the purification.Mentionable, the new buffer was not found by trial-and-error approaches.We searched for literature dealing with homologous proteins, especially for established purification protocols.This literature search revealed the new buffer, illustrating that not every step towards a protein structure determination must be a trial-and-error process.Another example for the influence of the buffer composition was published bei Mavaro et al. (Mavaro, Abts et al. 2011).Instead of the buffer agent, the ionic strength of the buffer was the crucial determinant.Purification of the protein in low-salt buffer resulted in an inhomogenous protein sample containing a mixture of aggregates, dimers and monomers without biological activity.However, a simple change to high-salt buffer allowed the purification of homogenous dimeric protein, that was able to bind its substrate.

Overcoming protein instability
In the previous sections different strategies were mentioned to enhance the stability and the homogeneity of purified proteins and in all cases the buffer composition was the solution.Still, the essential question how to determine the optimal buffer to make a protein feel happy in solution is not answered?Some rationales and experiences are listed above: different elution strategies for IMAC purifications, the usage of frequently used buffer agents and a literature research for established purification protocols of related proteins.However, in many cases these approaches do not solve the problems occuring during the purification.But, is there a general methodology to overcome the problems?Unfortunately, the answer is as frustrating as challenging -there is not a general panacea around for the right buffer composition of a protein.If a new buffer needs to be found, trial-and-error approaches have to be applied.A lot of different parameters are influencing the state of a protein, i. e. the buffer agent, the salt concentration, presence of metal ions with different valences, the hydrophobicity, and even the temperature of the buffer.The analysis of the protein in different buffers can be done by SEC and/or light scattering experiments.However, screening of all the different variables is very labor-and cost-intensive, and timeconsuming, moreover only combinations of two or more additives might be sufficient to enhance the solubility and homogeneity of the protein.Therefore high-throughput methods are needed that handle a lot of different conditions simultaneously using as few as possible protein sample.

Buffer screen -Enhancing the solubility
Many publications are available suggesting methods for a solubility screening to allow the crystallization of initially inhomogeneous, aggregating protein samples (Jancarik, Pufan et al. 2004;Zaitseva, Holland et al. 2004;Collins, Stevens et al. 2005;Sala and de Marco 2010;Schwarz, Tschapek et al. 2011).In all of these methods aggregating protein samples are mixed with commercially available crystallization screens incubated for a period of time, and analyzed for precipitation visually using a light microscope.Screening conditions resulting in no precipitations are analyzed upon their composition, and protein samples are further examined with respect to their solubility and homogeneity under these conditions by SEC or light scattering experiments.This technique allows high-throughput screening in a 96-well format, where an automated pipetting system mixes only 50-200 nL of protein solution with 50-200 nL of buffer solutions to minimize the needed protein sample and increase the screening efficiency.Several buffer screens are commercially available that cover many different buffer agents, salt concentrations and other buffer parameters (i.e. from Hampton Research, Molecular Dimensions, Sigma, Jena Bioscience, Qiagen).After a solubility screening was applied, we were able to stabilize a previously unstable protein at concentrations above 3 mg/mL (see above "Protein precipitations during IMAC" and Figure 2D) at concentrations of up to 100 mg/mL for weeks (Schwarz, Tschapek et al. 2011).Typically, the new buffer (50 mM citrate, 50 mM LiCl 2 , pH 6,00) should be used during the entire purification procedure starting with cell lysis.In the described case, the new buffer contains citrate, which is incompatible with an IMAC purification.Therefore the protein was immediately mixed with the new buffer after the elution of the IMAC column.

Size-exclusion chromatography versus light scattering experiments
Size-exclusion chromatography (SEC) and light scattering experiments (LS) are very helpful tools to analyze the homogeneity (Collins, Stevens et al. 2005) and the molecular mass of proteins; however both of them have advantages and disadvantages compared to each other.In SEC experiments proteins are separated based on their hydrodynamic radius by partitioning between a mobile phase and a stationary liquid within the pores of a matrix.All SEC columns are characterized by the volumes V 0 , the liquid volume in the interstitial space between particles, V i , the volume contained in the matrix pores and V T , the total diffusion volume (V 0 + V i ) (Regnier 1983a;Regnier 1983b).In dependency of the hydrodynamic radius molecules are eluting at specific retention volumes in between V 0 and V T with big molecules eluting first.After a calibration of a SEC column with proteins of known molecular weight (i.e. Sigma-Aldrich, "Kit for Molecular Weights") the molecular mass of the protein of interest can be roughly estimated; the elution volume is correlated to the log 10 of the molecular weight (therefore, the hydrodynamic radius is considered to be proportional to the molecular weight).However, many extraneous mechanisms such as adsorptive, hydrophobic and ionic effects are further limiting the correlation between the retention volume and the molecular mass giving sometimes rise to wrong estimations.Light scattering (LS) experiments can be applied to overcome these disadvantages and investigate the exact molecular weight of the protein sample.The rayleigh scattering of particles of monochromatic light depends directly on the molar mass of the particle.If you know the exact number of particles you can calculate the average molar mass of these particles.This technique is very powerful when used online after separation of the protein depending on their hydrodynamic radius, meaning SEC.This technique is always superior to normal SEC but requires special equipment and especially more time.However, if the protein fold is not really globular or other effects occur (see above: ionic, hydrophobic, etc.) assumption on size and oligomeric state based on SEC is not possible at all.For protein crystallization information about monodispersity, which can be provided by such an experiment, is an additional benefit.

Analysis of the homogeneity -High-throughput methods
Despite the development of various sophisticated methods, a bottleneck of homogeneity screening is high-throughput analysis.As mentioned above, proteins need to be analyzed by SEC and/or LS experiments after visual read-out of the protein-buffer droplets.Therefore, fluorescence-based solubility screens were developed that allow the high-throughput analyzes of many samples in a 96-well format (Ericsson, Hallberg et al. 2006;Alexandrov, Mileni et al. 2008;Kean, Cleverley et al. 2008).All these assays use fluorophores as reporters of the protein state.A suitable fluorophore is, for example, Sypro Orange, which exhibits different fluorescence properties as a function of its environment.This dye is almost dark in hydrophilic environment, however, after binding to hydrophobic molecules, it emits light at 570 nm.In inhomogenous and unfolded protein samples hydrophobic amino acids are exposed to the surface of proteins (Murphy, Privalov et al. 1990).An increase in the fluorescence signal of Sypro Orange correlates therefore with unfolding events of proteins.The homogeneity screening can be performed in basically two ways: temperature-or timedependent.For the first setup the protein sample is heated gradually in distinct steps (i.e. 1 °C) and the emission is monitored at 570 nm.Hereby, a "melting" temperature is determined, which is characterized by 50% fluorescence of the maximal fluorescence at the highest temperature; the higher the melting temperature, the higher the stability of the protein (Ericsson, Hallberg et al. 2006).Secondly, the protein sample is incubated at a specific temperature (i.e. 40°C) and the fluorescence is measured for a period of time.The "half-life" time, at which 50 % fluorescence of the maximum fluorescence in one sample is detected, can be compared to all buffer conditions.In Figure 4 an example of the timedependent approach is shown.Here, the protein is incubated in different buffers with various salt concentrations.The emission of Sypro Orange is recorded each minute at 570 nm.An analysis of all time-dependent fluorescence plots indicates that the protein is most stable in buffers containing 125 mM NaCl but unfolds fast in 1 M ammonium sulfate.These assays result in qualitative indications about a favourable environment of proteins that enhance the stability.Ericsson et al. proved the concept of this method by applying it to different proteins (Ericsson, Hallberg et al. 2006).The stability optimization yielded a twofold increase in initial crystallization leads.Moreover these assays enable the search for putative ligands of the protein.Upon binding of a substrate in the binding pocket or an inhibitor, the stability of the protein increases, which can be detected experimentally.

Protein crystallization: Introduction
Protein crystals suitable for X-ray diffraction experiments and usable for subsequent structure determination are normally relatively large with a size of at least 10 to 100 m.In contrast to crystals of mineral compounds, protein crystals are rather soft and sensitive to mechanical stress and temperature fluctuations.These properties are due to weak interactions between single proteins within the crystal, their high flexibility as well as the size of the macromolecules.The periodic network of building blocks is held together by dipole-dipole interactions, hydrogen bonds, salt bridges, van der Waals contacts or hydrophobic interactions.All of them have binding energies in the low kcal/mol range.Especially the limited number of crystal contacts and their directionality are the largest difference to the high interactions generally observed in salt crystals.An example of the interactions within a protein crystal is shown in Figure 5.This picture highlights the main pitfalls in protein crystallization.A protein is a highly irregular shaped and flexible macromolecule which allows weak and stinted interactions at very specific locations of its surface.All vacuity is filled with buffer, in general not contributing to any kind of interactions between the protein molecules.Figure 5A shows a protein of around 30 kDa, which crystallizes in a rather small unit cell (shown in black).Only one protein monomer is located in the asymmetric unit of the unit cell, the other shown monomers represent symmetry related proteins.Figure 5B highlights the three-dimensional packing of protein molecules within a crystal.The flexibility as well as the other mentioned characteristics of proteins are responsible for the problems occuring during crystallization trials and despite extensive efforts not every protein is suitable for crystallization.If one cannot generate crystals one has to move back several steps and change the properties of the protein, e. g. surface properties by mutation of single amino acids, truncation of the protein or sometimes only changing buffer compositions that result in a more suitable protein for crystallization (see Figure 1 and also below).There are several prediction servers available that help choosing the 'right' protein and modification (Linding, Jensen et al. 2003;Goldschmidt, Cooper et al. 2007).However, protein crystallization still remains an empirical approach, sometimes called voodoo, while crystallography is science.

Phase diagram
The conditions or protocols for obtaining good crystals are still poorly understood and despite all progress and efforts protein crystallization is a trail-and-error approach.However, a step towards a better understanding of crystal growth can be achieved by analyzing the phase diagram of a protein-water mixture.The phase diagram is a simple illustration to help understanding how protein crystals are formed.Mostly, it is shown as a function of two ambient conditions that can be manipulated, i. e. the temperature and the concentration.Three-dimensional diagrams (two dependent parameters) have also been reported (Sauter, Lorber et al. 1999) and even a few more complex ones have been determined as well (Ewing, Forsythe et al. 1994).Figure 6 shows a schematic phase diagram for a protein solution as a function of protein concentration and precipitant concentration.The phase diagram is broken down into four distinct zones (Rosenbaum and Zukoski 1996;Haas and Drenth 1999;Asherie 2004): 1. Undersaturated zone: Under these condition the protein will stay in solution as neither the concentration of the protein nor of the precipitant is high enough to reach supersaturation.2. Precipitation zone: Is the protein concentration or the precipitant concentration too high, the protein precipitates out of solution; this kind of solid material is not useful for crystallographic studies.
3. Labil zone: This is the most important configuration of the two parameters, as nucleation and initial crystal growth take place under these conditions.4. Metastable zone: After initial crystals are formed and start growing in the labil zone, protein concentration decreases in the drop and the metastable zone will be reached.
Here the crystal can grow further to its final maximum size.
Fig. 6.A basic solubility phase diagram for a given temperature (adapted from (Rupp 2007).
The curve separating the undersaturated zone from the supersaturated one is called solubility curve.If conditions are chosen below the solubility curve, the protein will stay in solution and never crystallize.This means when a protein crystal is placed in a solvent, which is free of protein, it will start to dissolve.If the volume of the droplet is small enough it will not dissolve completely: it will stop dissolving when the concentration of the protein in the droplet reaches a certain level.At this concentration the crystal loses protein molecules at the same rate at which protein associate to the crystal -the system is at equilibrium.Determination of the solubility of the protein of interest might be a helpful information at the beginning of crystallization experiments.This can be done in a twodimensional screen varying for example ammonium sulfate concentrations as well as the protein concentration.

Crystallization techniques
Crystallization is a phase transition phenomenon.Protein crystals grow from a supersaturated aqueous protein solution.Varying the concentration of precipitant, protein and additives, pH, temperature and other parameters induce the supersaturation.However, as mentioned before, prediction of this kind of phase diagrams is a priori impossible.Protein crystallization can be divided into two main steps: 1. Generating initial crystals: 'Searching the needle in a haystack' 2. Empirical optimization of these crystallization condition The first step is mostly based on experiences from other crystallization trials with different proteins.Nowadays several supplier offer crystallization screens that contain solutions for initial experiments that were used successfully in the past for crystallization trials (Jancarik and Kim 1991), so-called "sparse matrix screens".There are also some trials around to use more systematic approaches (Brzozowski and Walton 2000) to get more information about solubility prior and simultaneous to crystallization (incomplete factorials, solubility assays).Both kinds of screens can be applied to different crystallization techniques.Fig. 7. Crystal optimization.First steps in crystal optimization are shown.Initial protein crystals look weak and fragile, after screening around this initial buffer composition crystal evaluation by eye results in less fragile, homogeneous looking crystals.However, diffraction quality was poor.Therefore an additive screening was performed that resulted in a different crystal form.These crystals finally were able to diffract X-rays to a reasonable resolution.
A lead/hit in that initial step might not be a 'real' crystal rather than a crystalline precipitate or just phase separation.In the next step, fine-tuning the buffer composition further optimizes this hit.Varying pH, salt concentration, type and concentration of precipitant and protein concentration are expected to yield larger and hopefully also better-diffracting crystals.In this step, the chemicals used are much more defined and therefore it is a more systematic than empirical screening (see Figure 7).

Vapor diffusion
The most popular and simplest technique to obtain protein crystals is the vapor diffusion method either in the sitting or hanging drop variant (see Figure 8).For both a defined volume (mostly < 1µl) of protein solution is mixed with an equivalent volume of screening solution and then equilibrated against the original precipitant/screening concentration.During this equilibration, the vapor pressure of the solution rises as the protein crystallizes (protein in solution lowers water activity) while the water evaporates to maintain equilibrium, which causes the precipitant concentration to rise.Therefore, if the crystal growth is sensitive to the precipitant concentration, vapor diffusion can rapidly force the mixture to unstable conditions where growth and nucleation are too rapid.This is the main disadvantage of vapor diffusion: Growing large crystals might be problematic!

Micro batch method
In this set-up the protein solution is mixed with screening solution at concentrations required for supersaturation right at the beginning of the experiment.Typical drop sizes of micro batch experiments ranges from 1-2 µl.The drop is then covered with oil, which acts as an inert sealing to protect the drops during incubation from evaporation (see Figure 8).

Micro dialysis
Dialysis is another way to change the buffer composition and increase its concentration in the crystallization experiment gradually (see Figure 8).Micro-dialysis buttons are exposed to different screening buffers.This method requires rather high amounts of protein but might yield large crystals.After obtaining initial crystal hits in a commercial screen the tough part of crystal optimization starts.By varying pH, salt concentration, temperature, precipitant concentration or protein concentration these initial crystals should be reproduced and become larger, more regular shaped or are simply growing faster.A further improvement of crystal quality might be achieved by the addition of small amounts of so called 'additives'.At this point basically each chemical compound might be sufficient to improve the crystal quality.Luckily, there are some preferable working additives, which have been proven to produce better crystal in more than one case.Especially compounds that are known to reduce undirected interactions in proteins like organic solvents, i. e. DMSO or phenol, or detergents and reducing agents are very often used at this stage and helpful to force more homogeneous well diffracting crystals.

Crystal nucleation
There are two fundamental steps during protein crystallization: Nucleation and crystal growth.If one cannot obtain single crystals of adequate quality for analysis, this is generally a consequence of problems associated with the growth phase (see above).But failure to obtain any crystals at all or failure to obtain single, supportable nuclei reflects difficulties in the nucleation step.Therefore control of nucleation is a powerful tool to optimize protein crystals or sometimes it is the only way to get crystals at all.Nucleation can take place either homogeneous meaning in the bulk of the solution, when the supersaturation is high enough for the free-energy barrier to nucleus formation to be overcome or heterogeneous mostly by solid material in the solution.This can also occur even when the supersaturation is not achieved.Therefore in order to control nucleation one has to work with highly clean solutions to avoid nucleation by the second mentioned possibility.The nucleation zone can be bypassed by insertion of crystals, crystal seeds or other nucleants to the protein/precipitant mixture.Addition of crystals or tiny fragments of crystals is called seeding.This method is then subdivided into macro-and micro-seeding dependent on the size of the nucleant added.In macro-seeding experiments one single, already well-formed but small crystal is placed into a new crystallization solution at lower saturation.Microseeding in contrast requires small fragments of a crystal or almost invisible microcrystalline precipitate.These 'seeds' are then transferred into a fresh crystallization solution either by a seeding wand which is dipped into the microseed mixture to pick up seeds and then touched across the surface of the new drop or by a animal whisker or hair that is stroked over the surface of the parent crystal to trap the nuclei and then is drawn through the new drop.As this method also enhances the speed of crystal growth it can be used with sensitive substrate that undergo decomposition over time.Oswald et al. proved this in 2008 by solving the structure of ChoX from Sinorhizobium meliloti in complex with a highly hydrolyzing substrate, acetylcholine (Oswald, Smits et al. 2008).In classical vapor diffusion experiments crystals appear after four weeks but data showed only little electron density in the ligand-binding site and turned out to result from a choline bound instead of acetylcholine.Hydroxylation was favored due to the relatively long time for crystal growth but also because of an acetic pH in the crystallization set-up.To circumvent these problems accelerated crystal growth was required.In this case micro-seeding results in crystals suitable for data collection in less than 24 hours.Recent years more effort in nucleation control yielded in fancy materials that can be used as nuclei for crystals.These methods use the second way of nuclei formation, as a solid material is introduce into the crystallization solution as an 'universal' nucleant (Chayen, Saridakis et al. 2006).There have been several substances that have been tried more or less successful.Some have been useful for individual proteins, but mostly they were not applicable in general (McPherson and Shlichta 1988;Chayen, Radcliffe et al. 1993;Blow, Chayen et al. 1994). In 2001, Chayen et al. proposed the idea of using porous silicon whose pore size is comparable with the size of a protein molecule.In theory such pores may confine and concentrate the protein molecules at the surface of the silicone and thereby encourage them to form crystal nuclei (Chayen, Saridakis et al. 2001).These nucleants have made it to commercial availability (www.moleculardimensions.com) and have proven to be suitable for different kinds of proteins and even membrane proteins that have not been possible to crystallize before formed nice crystals in the presence of these nucleants.

Cryoprotection
Exposure of a protein crystal at room temperature results in dramatic radiation damage due to radicals formed by the ionizing X-ray photons.To reduce that harmful disintegration of the protein crystal the crystal is cooled to 100K with the help of liquid nitrogen (Low, Chen et al. 1966;Hope 1988;Rodgers 1994;Garman 1999).However, it is common for the cooling process to disrupt the crystal order and decrease diffraction quality.Thus, the crystal must be cooled fast so that the water in the solvent channels is in the vitreous rather than in the crystalline state at the end of this procedure.As for pure water this cooling has to take place very quick (10 -5 s, (Johari, Hallbrucker et al. 1987), some water molecules can be replaced by a cryoprotective solution prior to cooling (Juers and Matthews 2004).This exceeds the time window for up to 1-2s (Garman and Owen 2006) however, finding a good 'cryoprotectant' for a special protein crystal again involves substantial screening.Once flash frozen in liquid nitrogen, the crystal must be kept below the glass transition temperature of the cryobuffer at or below 155K at all times (Weik, Kryger et al. 2001).

What can you do when all efforts did not succeed in crystals? 4.5.1 Buffer composition -Again!
The choice of the right buffer used for crystallization experiments is very crucial.As shown above, every protein needs its own buffer composition to feel kind of happy in this aqueous artificial environment.Especially as high protein concentrations (>10mg/ml) are required for crystallization, one might has to test several buffer compositions again (see also Figure 1).As a rule of thumb you should obtain around 50% of clear drops immediately after mixing protein and buffer solution.If you detect drastically more precipitation in your drops you should think first about lower protein concentration but of course secondly about changing your buffer system again.

How to obtain a rigid protein suitable for crystallization?
To overcome the problem of flexibility of some regions in the protein addition of ligands is often a very powerful tool to fix the protein in a single conformation that is more favorable for crystallization.A good example for this strategy is the crystallization of so-called substrate binding proteins (for a recent review see (Berntsson, Smits et al. 2010)).These proteins catch their substrate in the periplasm of bacteria or on the outer membrane of archaea and then deliver it to their cognate transport system located in the membrane.The mechanism of substrate binding is quite well understood.These binding proteins all consist of two domains, which rotate towards each other during the binding event.In solution without substrate they are quite flexible and NMR-studies proved a equilibrium between open and closed conformation (Tang, Schwieters et al. 2007).Analysis of all available structures for this class of proteins showed that more than 95% were crystallized with a ligand bound (Berntsson, Smits et al. 2010).Thus, a stabilization of the two domains seems to simplify crystal contact formation dramatically.Although people always want to obtain a functional conformation of the protein in their crystal structure, it is sometimes helpful to think about how to stop the protein from doing its job.A non-functional protein is in general less flexible and fixed in one conformation.One example for successful implementation of this strategy is the crystal structure of NhaA from Escherichia coli solved in 2005 (Hunte, Screpanti et al. 2005).Here, Hunte et al., downregulated the protein activity by working at an acidic pH of 4.Although the protein shows almost no activity at this pH the structure reveals the basis for mechanism of Na+/H+ exchange and also its regulation by pH could be understood.

Rational protein design for crystallization: Surface engineering
The first example of rational protein design that yielded a good diffracting protein crystal is given by Lawson et al. in 1991(Lawson, Artymiuk et al. 1991).They compared amino acids involved in crystal contact formation of the rat ferritin protein L. (which is highly homologous to human ferritin H, the target protein) with the amino acids present at that position in human ferritin H.A replacement of Lys86, found in the human sequence, with Glu, which occurs in rat, recreated a Ca 2+ binding bridge that mediates crystal contacts in the rat ortholog.As this method was successful for several other proteins (McElroy, Sissom et al. 1992;Braig, Otwinowski et al. 1994;Horwich 2000), a general protocol was required.

GH T S S     
As the enthalpy values of intermolecular interactions in a crystal lattice are rather small (see above), crystallization is very sensitive to entropy changes of both protein and solvent.The formation of ordered protein aggregates carries a negative entropy term.This can only be overcome by positive entropy from the release of water bound to the protein.
However, large hydrophilic residues (e.g.lysines, arginies, glutamates, glutamines) exposed on the protein surface need to be ordered.Since they are rather flexible this can cause problems.This can be overcome by mutating large amino acids into smaller ones, for example alanines.Among these large amino acids lysines and glutamates play a particular role, as they are always (with only very few exeptions) located on the protein surface (Baud and Karlin 1999).Both lysines and glutamates are typically disfavored at interfaces in protein protein complexes (Lo Conte, Chothia et al. 1999), therefore it is rather straight forward to assume that lysine and glutamate to alanine mutants are good targets for protein crystallization if wildtype protein hardly forms crystals.However this also means that you have to go several steps backwards on road to a protein structure determination (see Figure 1).

Affinity tag removal: Philosophic question???
Another variant in protein crystallization nowadays is the affinity tag used for purification of the desired protein.The decision about position and choice of the affinity tag are mostly made at the beginning of the long way to a crystal structure (see Figure 1).However, it becomes crucial again at the crystallization step.In general most people like to remove the tag before crystallization to prove a physiological conformation.But, there are examples where the tag played a pivotal role in crystallization (Smits, Mueller et al. 2008a).The crystal structure of the octopine dehydrogenase from Pecten maximus is shown in Figure 9 (Smits, Mueller et al. 2008b), with the interactions sides/crystal contacts highlighted in green.
In Figure 9A contacts look quite similar to that presented in Figure 5. However when having a closer look on the his-tag, you recognize that it is located in a cavity formed by another monomer of that protein.In that cavity it can perform several hydrogen bonds with amino acids from the other monomer resulting in a very strong interaction which yields good quality crystals.

Crystallization using antibody fragments
A number of ways to stabilize proteins for crystallography have been developed, for example genetic engineering, co-crystallization with natural ligands and reducing surface entropy (see above).Recently, crystallization mediated by antibody fragments has moved into the focus of crystallographers especially to obtain crystals of membrane proteins (Ostermeier, Iwata et al. 1995;Hunte and Michel 2002).Membrane protein crystallization is even tougher compared to soluble proteins, because of the amphipathic surface of the molecules.As they are located in the lipid bilayer most of their surface is hydrophobic and must be covered to keep them in solution.This is maintained by detergents.The detergent micelles cover the hydrophobic surface and therefore this area is no longer available to form crystal contacts.Crystal contacts can only be formed by the polar surfaces of these proteins.As many membrane proteins contain only relatively small hydrophilic domains, a strategy to increase the probability of getting well-ordered crystals is required.Antibody fragments can play this role.They can be designed for binding at specific regions in the protein and then function as additional polar domain in the membrane protein complex (for example see (Ostermeier, Iwata et al. 1995;Huber, Steiner et al. 2007).

Conclusion
For what reason do we effort so much work on good quality crystals?Single good quality crystals constitute an essential prerequisite for structural investigations of biological macromolecules using X-ray diffraction.The harder one works on crystal quality the easier the determination of a reasonable atomic model of the molecule of interest becomes.The vast majority of problems encountered in crystal structure determination can typically be traced back to data-quality issues caused by crystal imperfections.Consequently, although primary focus of structural biology is on the macromolecule that makes up a crystal, there is also considerable interest in the physical properties, nucleation and growth of the crystals themselves.Statistics of various Structural Genomics Centers proved that protein crystallization is still despite all the progress in the technology of crystallization robotics is still a rather tough field in biological science.Success rate ranges for small prokaryotic proteins from 10-30% and decreases dramatically to a few percent for human proteins.The struggle obtaining crystals for protein structure determination is justified.After all efforst looking at electron density and subsequent the protein structure is still one of the most intriguing as well auspicious parts in structural biology

Fig. 2 .
Fig. 2. Elution fractions of an IMAC.The protein was eluted via a linear imidazole gradient from 10 to 500 mM and the absorption at 280 nm was recorded.The elution fractions were collected and photographed.A: IMAC chromatogram of the his-tagged protein.Elution fractions containing the desired protein (indicated by a bar) are collected and shown in B -D. B and C: Elution fractions of the protein in 50 mM Tris-HCl, 150 mM NaCl, pH 8.0 immediately and 10 min after the elution, respectively.D:The elution fractions were immediately mixed in a 1 to 1 ratio with a buffer that enhances the protein stability (50 mM citrate, 50 mM LiCl, pH 6.00) evaluated during a solubility screen.

Fig. 3 .
Fig. 3. Size exclusion chromatograms (UV 280nm) of proteins in different buffers.A: The homogeneity of a protein was analyzed in two different buffers; continuous line: 50 mM Tris-HCl, 150 mM NaCl, pH 8.00; dotted line: 20 mM Hepes, 150 mM NaCl, pH 7.00.B: The protein was eluted of the IMAC column either with imidazole (continous line) or with histidine (dotted line), concentrated and applied to the SEC.

Fig. 4 .
Fig. 4. Time-dependent stability optimization screen using Sypro Orange as reporter.The protein is diluted 1:50 into each test buffer containing Sypro Orange, excited with 490 nm and the fluorescence at 570 nm is measured for 60 minutes automatically with a PLATE READER (Fluorostar, BMG Labtech).Normalized fluorescence is plotted against the time.

Fig. 5 .
Fig. 5. Example of the packing within a crystal.A: The unit cell is shown in black, crystal contacts are highlighted with purple circles and lines.B: Three-dimensional crystal packing of a different protein.The unit cell as well as one protein monomer are depicted in green.

Fig. 8 .
Fig. 8. Protein crystallization techniques.Schematic representation of a) vapor diffusion, b) micro batch and c) micro dialysis crystallization techniques widely used for crystal growth (adapted from (Drenth 2006)).

Fig. 9 .
Fig. 9. Crystal contacts in OcDH protein.A: Overall view on two monomers.Surface Crystal contacts are highlighted in the green circles.B: Zoom in on the His-tag of one monomer.The his-tag of one monomer in the crystal structure is located near the binding site in a deep cavity formed by the other monomer.Therefore it is able to form several hydrogen bonds (highlighted in green) with side and main chains of the other protein but also with the ligand bound in this binding site (orange).

Table 1 .
Overview of expression systems.Summarized are the advantages and disadvantages.
The concept Derewenda et al. proposed in 2004 is based on the general equation for the free energy that drives protein crystallization: