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Protein Crystallisation in Action

Next: 2 Experimental methods and Up: Protein Crystallisation in Action Previous: List of Figures


1 Introduction to protein crystallisation

1.1 General principles of crystallisation theory

The word 'crystal' is derived from the Greek word 'krustallos' (clear ice). Like ice, crystals are physically homogeneous solids; many of them have a transparent glittering appearance and a well-defined geometrical shape, with regular faces and sharp edges. Crystals exhibit a high degree of internal three-dimensional order and a definite, although not necessarily stoichiometric, overall chemical composition [10]. The high degree of internal order means that the array of atoms, ions, molecules, or molecule assemblies is ordered over many atomic dimensions. Short-range order means that the order exists only within a few atomic dimensions. The disruption of the long-range internal order causes the material to lose its crystalline properties.

The crystallisation process of molecules of any compound from its solution represents a reversible equilibrium phenomenon, driven by the minimisation of the free energy of the system [20]. A solution, in which the molecules are fully solvated, corresponds to the system at equilibrium; in other words its free energy is minimised. If more molecules are added to the solution, the system undergoes internal changes until the point is reached where there is insufficient solvent to maintain full hydration of the molecules. Under these conditions (the so-called 'supersaturated state') the system is no longer at equilibrium. Therefore, it will be thermodynamically driven toward a new equilibrium state with a corresponding new free energy minimum. Individual molecules lose rotational and translational freedom by forming many new stable non-covalent chemical bonds, thus minimising the free energy of the system. Crystallisation is known to lower the free energy of proteins by approximately 3 - 6 kcal/mole relative to the dissolved state in solution [6]. This aggregation results in partitioning of the molecules between soluble and solid phases. The solid phase can appear in a state of either amorphous precipitate or crystal nuclei. Amorphous precipitates are usually more favourable kinetically, and so they tend to dominate the solid phase and inhibit crystal formation.

The principles of crystal growth have been the subject of intense investigation for a number of years. As a consequence, the theoretical and practical aspects of crystallisation of molecules like salts or small organic compounds are nowadays well established. Over the past decade these aspects have been proven to be similar, to some extent, to those related to the crystallisation of macromolecules, like proteins, DNA and RNA (for the more recent review see: [14]). The basic strategy is to bring the system into a state of limited degree of supersaturation by modifying the properties of the solvent through equilibration with a precipitating agent, or by altering some physical properties such as temperature, etc. Three stages of crystallisation are common to all systems: nucleation, growth, and cessation of growth.

The nature of the crystal nuclei is largely a mystery. It is not known whether they are initially ordered, ordered after a certain degree of internal re-arrangement, or whether they form by the coalescence of random subnuclear clusters or by strict monomer or oligomer addition. It is believed, though, that in the crystal nuclei, molecules show the tendency to make interactions with one another in all three directions in a periodic manner [16]. A different scenario occurs when the amorphous precipitates form, as they are characterised by random intermolecular interactions. These unstructured aggregates tend to form over a much faster time scale than regular crystals. The formation of crystal nuclei from supersaturated solutions does not necessarily imply the subsequent formation of macroscopic crystals. In fact, the nucleus must first exceed a specific size, called the critical size, defined by the ratio between the surface area of the nucleus and its volume [1]. Once the critical size is exceeded, the nucleus is capable of further growth, given the availability of protein molecules in solution and suitable physical-chemical experimental conditions. If the nuclei are smaller than the critical size, spontaneous dissolution will occur.

The growth of macroscopic crystals from the initial nuclei is strongly influenced by diffusion and convection effects. Feher and Kam, by means of ultraviolet microscopy techniques, have been able to demonstrate that the regions in the near proximity of the growing crystal have a lower protein concentration than the rest of the solution [8]. The formation of these zones has the effect of producing density gradients in the solution. These, in turn (under the effects of gravity), result in the formation of convection currents, which dominate the rate of simple diffusion and could unfavourably affect the crystal growth. Crystal growth in effective zero gravity may be used to remove convective and sedimentary effects [5]. Crystal growth is also dependent on the nature of the growing crystal surface. The addition of molecules to a rough surface normally requires less energy than the growth from a smooth surface [20].

A classical explanation of crystal nuclei formation and growth is given by the two-dimensional solubility diagram shown in figure 1. The solubility curve divides the concentration space into two areas - the undersaturated and supersaturated zones. Each point on this curve corresponds to a concentration at which the solution is in equilibrium with the precipitating agent. These correspond to the situation either at the end of the crystal growth process from a supersaturated solution or to a situation when crystal dissolution occurs in an undersaturated solution. In the area under the solubility curve, the solution is undersaturated and the crystallisation will never take place. Above the solubility curve lies the supersaturation zone; here, for a given concentration of precipitating agent, the protein concentration is higher than that at equilibrium. Depending on the kinetics to reach equilibrium and the level of supersaturation, this region may itself be subdivided into three zones:

  1. The precipitation zone is where the excess of protein molecules immediately separates from the solution to form amorphous aggregates.
  2. The nucleation zone is where the excess of protein molecules aggregates in a crystalline form. Near the precipitation zone, crystallisation may occur as a shower of microcrystals, which can be confused with precipitate.
  3. A metastable zone; a supersaturated solution may not nucleate for a long period, unless the solution is mechanically shocked or a seed crystal introduced. To grow well-ordered crystals of large size, the optimal conditions would have to begin with the formation of a preferably single nucleus in the nucleation zone just beyond the metastable zone. As the crystals grow, the solution would return to the metastable region and no more nuclei could occur. The remaining nuclei would grow, at a decreasing rate that would help to avoid defect formation, until equilibrium is reached.

Figure 1: Crystallisation phase diagram. Schematic representation of a two-dimensional phase diagram, illustrating the change of protein molecules concentration against precipitating agent concentration. The concentration space is divided by the solubility curve into two areas corresponding to undersaturated and supersaturated state of a protein solution. The supersaturated area comprises of the metastable, nucleation and precipitation zones [7].
\begin{figure}{\centering\includegraphics{pics/01-phase-diagram.eps}\par }

Cessation of crystal growth can occur for a multitude of reasons. The most obvious is the decrease in concentration of the crystallising solute to the point where the solid and solution phases reach exchange equilibrium. In this case, the addition of more solute can result in continued crystal growth. However, some crystals reach a certain size beyond which growth does not proceed, regardless of solute concentration. This may be the result of either cumulative lattice strain effects or poisoning of the growth surface.

Poisoning of growing crystals occurs when foreign or damaged molecules are incorporated into the growing crystal face, resulting in successive defects, which interrupt the crystal lattice. An example of this might be the incorporation of a proteolytically damaged protein onto the face of an otherwise perfect protein crystal. If the damaged molecule is unable to form the same lattice contacts with newly added molecules, as would the perfect protein, then its incorporation will cause local defects in the growing crystal. As crystals grow larger, the likelihood of incorporation of defective molecules into the lattice increases. Sato and co-workers have used laser scattering tomography to visualise lattice defects in large orthogonal crystals of hen egg white lysozyme [18]. Their results demonstrated the occurrence of defects not only at the surface, but also within the bulk of the crystal itself. The poisoned surfaces can be removed by partially melting the crystal. This provides an undamaged surface, which is capable of further growth in the presence of more solute. A method for applying this technique to protein crystals has been designed by Thaller and co-workers [19].

1.2 Protein crystallisation philosophy

Although biological macromolecule systems share the same fundamental mechanism and pathways of crystal growth as small molecules, the magnitude of the underlying kinetics and thermodynamic parameters regulating the process differ dramatically. The major contributing factors include:

The other crucial difference between protein and small molecule crystallisation is that protein crystals usually require extremely high levels of supersaturation for nucleation. Although high levels of supersaturation are necessary to promote nucleation of protein crystals, these conditions are normally far from ideal for crystal growth. In fact, when supersaturation is achieved, the competition between crystal nuclei and amorphous precipitation becomes severe. Additionally, biomacromolecular crystals have to be grown from chemically rather complex aqueous solutions, that in turn increase the number of parameters that need to be controlled for successful protein crystallisation. Consequently, the optimal conditions of protein crystal growth are virtually impossible to predict a priori.

By far the most important factor in crystallisation is the purity of the protein sample. Ideally, a purity approaching 100 % is necessary for a successful protein crystallisation experiment. Any contamination, either by a small amount of co-purified proteins or by degradation products of the protein itself, can inhibit crystal formation or lead to contamination of the crystal lattice, and ultimately to poorer crystals. SDS PAGE may not be enough to verify the purity of a protein sample, so other methods like mass spectrometry, native PAGE or isoelectric focusing (IEF) have to be employed. Another fundamental precept of crystallisation is the absolute homogeneity of the protein sample. Since proteins are taken out from their natural environment and kept at concentrations much higher than the physiological ones, they might tend to form high molecular weight soluble aggregates. By means of light scattering analysis, the occurrence of such high molecular weight aggregates can be ascertained, and this problem can sometimes be overcome by the addition of detergent molecules. In one case, McPherson and co-workers reported an entirely new crystal form of a protein due simply to the addition of 0.1 - 1 % BOG (\( \beta \)-octyl glucoside, a mild detergent) to the crystallisation conditions [15].

As noted earlier, molecules crystallise from metastable supersaturated solutions. Chemical precipitants are the most widely used agents for achieving supersaturation of macromolecules in order to induce crystallisation. The influence of these compounds is mainly on the solvent, e.g. bulk water, rather than on the solute. Commonly used precipitants can be divided into several main categories: salts, high molecular weight straight chain polymers (e.g. polyethylene glycol), and organic solvents (2-methyl 2,4-pentanediol).

Salts are by far the most common precipitant used to crystallise macromolecules. Unfortunately, salts generally have the drawback of increasing the mean electron density of the crystallisation solution, which in turn has the effect of decreasing the signal-to-noise ratio of the crystallographic data. Salts also have a tendency to interact strongly with heavy atom compounds, making crystal derivatisation for MIR phasing more difficult.

Some proteins are poorly dissolved in pure water but do dissolve if a small amount of salt is added. By removing salt, the protein precipitates. This 'salting-in' effect can be explained by considering the protein as an ionic compound. According to the Debye-Hückel theory for ionic solutions, an increase in the ionic strength lowers the activity of the ions in the solution and increases the solubility of ionic compounds. Alternatively, one can regard the salting-in effect as the result of competition between charged groups on the surface of the protein molecule and the ions in the solution. In the absence of ions, the protein precipitates by Coulombic attraction between opposite charges on different protein molecules. If ions are added, they shield the charged groups on the protein and increase its solubility. A 'salting-out' effect occurs when the ionic strength is increased to the point where salt ions and protein molecules compete for solvent molecules i.e. water, to maintain their hydration layers and therefore their solubility. To fulfil their electrostatic requirements, the protein molecules begin to self-associate.
The use of high molecular weight linear polymers as precipitating agents was pioneered by Polson and co-workers, who tested a variety of polymers including polyethylene glycol, dextran, polyvinyl alcohol, and polyvinyl pyrrolidone [17]. Polyethylene glycol (PEG) was found to be the most effective both in terms of precipitating ability and cost effectiveness [12]. PEGs are produced in a variety of molecular weights, ranging from 200 to 1,000,000 Daltons. Like salts, PEGs compete with protein solutes for water and exert volume-excluded effects (which vary according to the length of the polymer). However, unlike salts, PEGs decrease the dielectric constant of the solution, which increases the effective distance over which protein electrostatic effects occur. Solutions of PEGs have mean electron densities roughly equivalent to water and do not generally interact in a damaging manner with heavy atom compounds. This makes them particularly well suited for macromolecular crystallisation. PEGs with molecular weights of less than 1,000 are typically liquids, and are generally used at concentrations above 40 % (v/v). PEGs with molecular weights above 1000 are generally solids and are used in the 5 - 50 % (w/v) concentration range. Buffering of high concentration (> 40 %) PEG solution with sodium citrate at concentrations above 100 mM tends to cause the formation of phase transitions and colour changes in the PEG solution. These changes are caused by cross-linking of PEG by the citrate, and thus should be avoided.
Organic solvents.
Organic solvents bind water molecules and divert them from their interaction with protein atoms. They also lower the dielectric constant of the medium, thus enhancing the Coulomb interactions between the molecules. These effects decrease the capacity of the system to fully solvate the protein molecules and reduce the electrostatic shielding, which in turn forces the protein molecules out of the solution. Some of these agents should be used at low concentrations since they often destabilise protein molecules at high concentrations.
The 2-methyl 2,4-pentanediol (MPD) is a small polyalcohol which has properties midway between those of low molecular weight PEGs and organic solvents. MPD functions as a precipitant by a combination of effects, including competition for water, hydrophobic exclusion of protein solutes, lowering of the solution's dielectric constant, and detergent-like effects. It is generally used in excess or in a concentration of 40 % (v/v). As with PEGs, the use of MPD as a precipitant produces a low level of electron density and does not generally interact with heavy atom compounds.
Although all of the chemical precipitants discussed above worked for one system or another, often their individual effects are too severe for crystallisation to occur. Instead, the protein solute leaves the supersaturated state by way of precipitation rather than crystallisation, or may form poor two-dimensional or very small numerous crystals. In such cases, combination of precipitants (i.e. salts and PEGs, MPD and salts, salts and organic solvents, etc.) may produce larger crystals (as happened with the HR1b and the NK1 proteins) and, occasionally, new crystal forms.

The most important factor in crystallising proteins, other than the chemical composition of the crystallisation solution, is its pH. The majority of the proteins tend to crystallise at physiological pH (around 7.5). However, some proteins (such as the HR1b domain described in this paper) have been crystallised at extreme values of pH. For some proteins, crystallisation occurs over a very broad range of pH with little variation in crystal morphology. However, it is far more typical for crystallisation to occur over a narrow range - typically within one pH unit. Crystal morphology, including various twinned forms, is often directly related to pH. In some cases, the pI of the protein is the pH at which the best crystals grow, so this should, if possible, be known in advance and tried as a condition.

Temperature is another important parameter in the crystallisation of a protein. The vast majority of protein molecules are crystallised either at 4  \( ^{\mathrm{o}}\mathrm{C} \) or 18 - 22  \( ^{\mathrm{o}}\mathrm{C} \) (room temperature). Low temperatures tend to limit degradation problems as well as bacterial growth. The solubility of proteins in salt solutions tends to increase at low temperatures, whereas in PEG and MPD solutions, protein solubility generally decreases with decreasing temperature. By increasing or decreasing precipitant or protein concentration, crystallisation should, at least in theory, be possible at either room temperature or 4  \( ^{\mathrm{o}}\mathrm{C} \), although the kinetics of crystallisation can be expected to vary in accord with temperature.

Chemical/biochemical modification of proteins is another factor, which may be used to change crystallisation conditions. The charge distribution on the surface of a protein plays an important role in dictating whether the protein crystallises or not. Thus, modification of surface charges by either chemical (derivatisation) or biochemical (mutagenesis) means can provide crystals where none were known before; or provide crystals in different space groups, possibly leading to a higher resolution data set.

1.3 Physical techniques of protein crystallisation

A number of techniques have been developed for bringing a protein solution into a supersaturation state (reviewed in: [7,14]). Among them, the following three methods are frequently used: micro-batch, vapour-diffusion and dialysis (figure 2). Although supersaturation of a protein solution could be achieved by means of each of these techniques, the underlying principles of these methods vary. In this study, micro-batch and vapour diffusion methods were extensively exploited during the crystallisation trials. Therefore, a detailed description of both these techniques will be presented below.

The micro-batch method is a variation of the simple batch crystallisation technique, where the concentrated protein solution is mixed with concentrated precipitant in a closed vessel to produce a final supersaturated concentration, which may eventually lead to crystallisation. This can be done with large amounts of solutions, and typically results in larger crystals owing to the larger volumes of solute present and the lower chance of impurities diffusing onto the face of the crystal. In the micro-batch technique, smaller volumes, as little as 0.5 ml, can be used. The protein sample and precipitant solution are dispensed into the well of a plate and the well is covered with paraffin oil to prevent evaporation (figure 2a). During the incubation period, the concentration of a precipitant agent remains constant since evaporation is limited and, therefore, the volume of the drop remains the same during the experiment. On the other hand, the concentration of the protein changes on formation of either crystals or amorphous precipitant. If the concentration of precipitant agent is chosen in such a way that the solution is in an undersaturated state, crystallisation will never occur. In terms of phase diagram, see figure 3a, this condition is indicated by point A. The protein will immediately precipitate if the starting point is located in the precipitation zone of the solubility diagram, point C. Therefore, only those points in the phase diagram (point B in figure 3a) which lie between the solubility and precipitation curves represent starting conditions for a successful crystallisation experiment. The main disadvantage of this method is that the equilibration occurs very rapidly, thus affecting the rate of crystal growth and consequently the quality of the obtained crystals. The second disadvantage is that the manipulation of the crystals from the drop covered by oil is very difficult. However, since the use of very small volumes of protein solution can be made, the micro-batch technique is quite useful as an initial screening method. This method was successfully employed for obtaining the initial NK1 protein crystallisation conditions. Although the evaporation of water from the drop covered by oil is negligible, it does occur, and therefore the 'life-time' of micro-batch trials is usually about 2 to 3 weeks.

Figure 2: Protein crystallisation techniques. Schematic representation of a) microbatch, b) vapour-diffusion and c) dialysis crystallisation techniques widely used in growing of protein crystals.
\begin{figure}{\centering\includegraphics{pics/02-xtal-methods.eps}\par }

Figure 3: Various crystallisation set-ups explained in terms of phase diagrams. Schematic representation of solubility phase diagram and correlation between protein and crystallising agent concentrations in a) batch, b) vapour-diffusion and c) dialysis crystallisation experiments. \( C_{ip}\) and \( C_{i}\) are the initial concentrations of protein and crystallising agent respectively, \( C_{fp}\) and \( C_{f}\) are their final concentrations.
\begin{figure}{\centering\includegraphics{pics/03-phase-diagrams.eps}\par }

The vapour-diffusion technique utilises evaporation and diffusion of water between solutions of different concentrations as a means of approaching and achieving supersaturation of protein macromolecules. Typically, the protein solution is mixed in a 1:1 ratio with a solution containing the precipitant agent at the concentration that needs to be achieved after vapour equilibration has occurred. The drop is then suspended and sealed over the well solution, which contains the precipitant solution at the target concentration, as either a hanging or sitting drop (figure 2b). The difference in precipitant concentration between the drop and the well solution is the driving force which causes water to evaporate from the drop until the concentration of the precipitant in the drop equals that of the well solution. Since the volume of the well solution is much larger than that of the drop (1 - 3 ml as compared to 1 - 20 ml), its dilution by water vapour leaving the drop is insignificant. During a vapour-diffusion experiment, the protein will start to concentrate from an unsaturated state (point A, concentration \( C_{pi} \)) to reach a supersaturated state (point B). As the first crystals appear the concentration of protein will decrease (figure 3b). The crystal will then grow until the concentration of the protein in the drop reaches the solubility curve (point C, at concentration \( C_{pf} \)).

Vapour-diffusion is the optimal technique when screening a large number of conditions. Furthermore, this method can be used to increase or decrease the concentration of protein in the equilibrated state, relative to its initial concentration. Varying the ratio between the protein and well solutions when the drop is initially set up does this. Since the drop equilibrates in such a way that its precipitant concentration matches that of the well solution, the final volume of the drop will always equal that of the initial amount of well solution mixed with the protein. Thus, if the protein solution is mixed in a 2:1 ratio with the well solution, the concentration of the protein at vapour equilibrium will be doubled, relative to its initial value.

The rate of vapour-diffusion can also be controlled, to a certain extent; it greatly depends on the surface area of the drop. By reducing the surface area we can slow the equilibration process dramatically. This can be achieved by means of the so-called 'sandwich drop' technique (see figure 2b). The volume of a hanging drop is limited to about 8 - 10 ml, since bigger volumes might cause the drop to fall out of the cover slip. This limitation is overcome in the 'sitting drop' technique (figure 2b).

Dialysis techniques utilise diffusion and equilibration of small precipitant molecules through a semipermeable membrane as a means of slowly approaching the concentration at which the macromolecule solute crystallises. Initially, the protein solution is contained within the dialysis membrane, which is then equilibrated against a precipitant solution. Equilibration against the precipitant in the surrounding solvent slowly achieves supersaturation for the solute within the dialysis membrane, eventually resulting in crystallisation. A dialysis membrane can be used to cover the opening of a dialysis button, allowing diffusion of the surrounding solvent into the solute through the dialysis membrane (figure 2c). Dialysis buttons themselves come in a variety of sizes from 7 ml to 200 ml. The protein solution at the start of the dialysis experiment is in an undersaturated state (figure 3c). The concentration of the precipitant agent slowly increases as its diffusion through the membrane takes place. Thus, the system goes from an undersaturated state into the metastable region through the point S on the solubility curve.

The advantage of dialysis over other methods is in the ease with which the precipitating solution can be varied, simply by moving the entire dialysis button or sack from one condition to another. Thus, the protein solution can be continuously recycled until the correct conditions for crystallisation are found. Although this method was tried during the crystallisation of the HR1b domain and the NK1 fragment, it was not used for primary crystallisation. The reason is that this method does not work with concentrated PEG solutions used for crystallisation of these proteins. The most obvious explanation is the PEG solutions tend to draw all the water out of the button or sack faster than dialysis take place across the membrane, thus resulting in precipitated protein.

1.4 Strategy of protein crystallisation

The crystallisation of an uncharacterised or even a well-characterised macromolecule is not nearly as straightforward as it might seem. In fact, there are a vast number of possible conditions, which must be analysed for their ability to trigger the crystallisation process. To illustrate this, consider an initial screen where only five different precipitants are to be used. Each of these five precipitants is set up at four different concentrations, and each of these groups is set up at a different pH, from 4 - 9 at one pH-unit intervals. At this point 120 conditions are to be tried. If two different temperatures and two macromolecule concentrations are tried for all these conditions, the number of these initial trials approaches 500. Needless to say, the more variables tested the greater the expense in terms of labour and the amount of purified protein.

In addition, the kinetics of nucleation and crystal growth varies in an apparently random fashion. Thus, a condition, which is viable for crystal growth but unstable for nucleation, may take days to months to produce crystals, whereas another condition might produce crystals within a matter of minutes. Worse yet, microscopic crystallisation might occur but go unnoticed due to its similarity in appearance to precipitated solute.

Various strategies have been implemented to screen possible initial conditions for crystallisation. These vary from somewhat rational approaches (screening at the pI) to highly regimented approaches (successive grid screening) to analytical approaches (incomplete factorials, solubility assays) to randomised approaches (sparse matrices). Each of these strategies has their own particular use based on whether the protein is unknown or well known. A short description of the various approaches with references is provided below.

Screening at the pI.
The pI of a macromolecule (the pH at which it has zero net charge) is its point of lowest solubility. Therefore, it is rational to assume this could be a point where crystallisation may occur. For example, in the case of the HR1b domain, the final pH of the crystallisation solution was within one pH-unit of the theoretically calculated pI. Screening at the pI may be done by dialysis against low concentrations of buffer (less than 20 mM) either at the appropriate pH or by the use of conventional precipitants.
Grid screening.
Grid screens are typically based on two-dimensional matrices, with two different parameters varied along the axes. Screening often results in an iterative process, starting with a course grid over a wide range of crystallisation conditions and ending with a very fine grid over a narrow range of a few selected parameters. This type of procedure could be implemented in an automated way by means of crystallisation robots [4]. Typically, the first screen conducted in this method consists of a matrix in which the precipitant concentration is tested versus the solution pH, as these are generally the two most important factors affecting the crystallisation of macromolecules. The result of the initial screen is then graded on the absence or presence, as well as the type, of precipitant, which can be flocculent (fluffy clouds), granular (amorphous grains), dendritic (string) or crystalline (from micro-needles to perfect crystals). A range of solutions is then prepared which includes and brackets the best initial conditions, and this procedure is repeated until the two-parameter grid converges on the best condition. This condition can then be used as an initial starting place to test additional factors (temperature, additives). This method works well if crystals appear early in the screening process, but otherwise becomes expensive in terms of protein due to the need to sample exhaustively a wide range of conditions.
Incomplete factorials.
Carter and Carter pioneered this approach as a "rational exploration of cause and effect relationships governing the crystallisation of proteins" [3]. The incomplete factorial approach is to take an initial set of approximately 20 conditions, and randomly assign combinations of these factors as individual experiments. The success rates of each experiment are then graded based on an arbitrary objective scale, and the effects of each of the twenty factors on the crystallisation trials are statistically evaluated [2]. Thus, the effects of factors such as pH, temperature, precipitating agent, and cations can be determined precisely with minimal subjective interpretation. These experiments are carried out in dialysis buttons so that the protein can easily be recycled (by dialysis against buffer to remove precipitant). Each experiment can be driven to completion by changing the precipitant concentration until either precipitation or crystals occur. In the two sets of trials reported by Carter and co-workers, final conditions yielding high quality crystals, sometimes in multiple space groups, were determined within 35 trials.
Sparse matrices.
Jancarik and Kim first described the use of a sparse set of conditions for initial crystallisation trials based on conditions, which were known to have crystallised proteins [9]. The Crystal Screen sold by Hampton is made-up of solutions suggested by Jancarik and Kim (see also: [13]). The idea behind sparse matrix trials is to provide a broad enough sampling of parameter space by random (or nearly random) combination of conditions to yield initial crystals, which then may be improved upon. This is also a very efficient method, when it works, in terms of consumption of the macromolecule, and therefore is preferable under conditions where the quantity of macromolecule is the limiting factor.
Sometimes the step of moving from microcrystalline precipitation to large crystals is unsuccessful. In this case a seeding technique can be utilised. Seeding is a method that physically separates the process of crystal nucleation and growth, so that conditions for crystal growth can be independently optimised (reviewed in: [7]). Although this method was not used during the course of this work, its brief explanation follows. The microseeding method encompasses transfer, by means of a fine glass rod, crystallisation nuclei (can be readily obtained from a microcrystalline precipitation) to a growth-promoting solution. Individual crystals having dimensions as small as 0.01 mm can be grown to larger sizes by macroseeding. In this procedure a single crystal is transferred into a fresh protein/precipitant agent solution once the crystal growth ceases. Before the transfer, the crystal is washed in a solution of low concentration so that its surface is etched by partial dissolution. The etching is needed to remove surfaces of the crystal already poisoned by impurity incorporation.

1.5 Pre-diffraction characterisation of a crystal

Once crystals have been obtained, tests should be carried out in order to prove whether they are protein or salt crystals. Small salt molecules are often present in crystallisation solution as an additive to the precipitating agent or as the precipitating agent itself. They sometimes produce crystals that are very difficult to distinguish from macromolecular crystals. The X-ray diffraction pattern is, of course, the most convincing proof of the nature of the crystals. In this way, the crystallographer will also immediately gain the information about crystal maximum resolution and crystal symmetry.

However, it should not be forgotten that simple biochemical analysis could clarify the question about the crystal's nature. A fairly straightforward way of checking the crystal's nature is to perform a crush test. The most striking difference between salt and protein crystals is that the latter exhibit very poor mechanical properties and high solvent content [11,20]. Consequently, protein crystals are always extremely fragile and very sensitive to external conditions. They will crush easily once touched with the tip of a needle, whereas salt crystals will only crush if a considerable force is applied.

The fragility of protein crystals is a consequence both of the weak interactions between macromolecules within the crystal lattice and the high solvent content. For these reasons, protein crystals have to be kept in a solvent-saturated environment to avoid dehydration. The high solvent content results in the presence of large channels within a crystal, which are filled with solvent. These solvent channels have useful consequences, since they allow the diffusion of small molecules or ligands inside the protein crystals. This feature is also exploited extensively for making heavy metal derivatives and checking the nature of newly obtained crystals. A dye-solution (Hampton Research) is used for the latter; it is usually added straight to the crystallisation drop with formed crystal. After several hours, if the crystal is of macromolecular nature, small dye molecules will penetrate through the solvent channels inside the crystal, causing it to discolour. As salt crystals are more densely packed and do not have extensive solvent channels, the change of the colour would not occur.

The methods described above could only tell if the crystal is of macromolecular nature or not. The crystallographer has to be sure that the crystal they have grown is not only protein crystal, but also the crystal of the protein of interest. One can harvest several large crystals, dissolve them and perform the SDS PAGE together with the sample of purified protein used for crystallisation. The behaviour of two protein samples can then be analysed and compared.

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