Advanced Biocrystallogenesis

Nowadays, X-ray crystallography is one of the most popular structural biology methods. Successful crystallization depends not only on the quality of the protein sample, precipitant composition, pH or other biophysical and biochemical param-eters, but also largely on the use of crystallization technique. Some proteins are difficult to be crystallized using basic crystallization methods; therefore, several advanced methods for macromolecular crystallization have been developed. This chapter briefly reviews the most promising advanced crystallization techniques and strategies as one of the efficient tools for crystallization of macromolecules. Crystallization in capillaries, gels, microfluidic chips, electric and magnetic fields as well as crystallization under microgravity condition and crystallization in living cells are briefly described.


Introduction
Macromolecular crystallization was invented accidentally in the late 19th century. The initial goal of crystallization processes was to purify and to prove the chemical purity of the examined chemical compound. One of the first achievements in macromolecular crystallization were crystals of hen-egg albumin at the end of the 19th century and crystals of insulin in the 1920s [1,2]. Since then, macromolecular crystallization has developed into a powerful tool for the threedimensional structure determination of nucleic acids, proteins as well as for larger macromolecular complexes.
A good-quality macromolecular crystal is needed in X-ray crystallography for obtaining a 3D model of corresponding nucleic acids, proteins, macromolecular complexes or larger biological assemblies, for instance, viruses or ribosomes. Nevertheless, macromolecular crystallization is a highly unpredictable process, which relies on a large number of chemical and biochemical parameters, such as the purity and concentration of the protein, type of precipitant, pH of the buffer, as well as on physical parameters, as temperature, time, pressure or vibrations, etc.
In order to crystallize the protein from the solution, the supersaturated state has to be reached using various crystallization techniques. Crystallization process can be divided into three stages -nucleation, crystal growth and termination. So-called phase diagrams typically illustrate these steps. For the investigation of the best conditions for crystal growth of the individual macromolecule, two general strategies are usually applied. Firstly, initial crystallization screening using commercially available screening kits is performed to explore the suitable conditions for crystal growth. Secondly, conditions yielding macromolecular crystals are systematically modified to allow the growth of the best-quality crystals adequate for X-ray diffraction analysis. Basic crystallization techniques, namely batch, vapour diffusion and free-interface diffusion are used for initial crystallization screening generally [3]. In addition, these techniques can be further modified to obtain as good-quality crystals as possible during the optimization of protein crystallization. However, some proteins cannot be easily crystallized and produced crystals in diffraction quality and thus initial hits need to be further improved. It is important to note that different crystallization methods screen the phase diagram from different points of view and hence affect the properties of the resulting crystals. Therefore, advanced biocrystallogenesis methods are recommended to be applied for screening or optimization of crystallization conditions. This chapter reviews different advanced methods and strategies as efficient tools for crystallization of macromolecules. During the past few years, the field of protein crystallography has significantly developed regarding instrumentation. Different technologies require different sizes of crystals, for instance homogeneous nanocrystals for XFEL or big size crystals for neutron diffraction [4,5]. This also drives the necessity to develop new and convenient methods for growing suitable high-quality protein crystals. For this purpose advanced crystallization methods and strategies have been developed.

Counter-diffusion
Counter-diffusion belongs to the non-equilibrium crystallization approaches. The principle of this method is based on the use of one-dimensional experimental chambers, including protein and precipitant solutions, resulting in slow mutual diffusion, in other words the slow mixing of the molecules of the solutions and therefore slow reaching of the supersaturation state [6]. During the diffusion, the solubility of the macromolecule is significantly reduced (Figure 1-(3)) [7].
The ideal concentration of the protein solution is advised to be higher (more than 4 mg/ml) and the concentration of the precipitant solution should be very high, nearly reaching the supersaturation state [8]. At the beginning of the experiment, the diffusion is faster and thus the amorphous precipitate is formed. Eventually, the protein concentration decreases at the liquid-liquid interface, which leads to the nucleation followed by the protein crystal growth [9].
Counter-diffusion method for protein crystallization and screening is popular since it screens a wide range of the phase diagram while carrying out only one experiment and increases the chance of hits at the same time.

Crystallization in capillaries
Crystallization in thin and long capillaries is one of the most used advanced crystallization technique, frequently applying counter-diffusion. Furthermore, capillaries can be used also in batch crystallization or vapour diffusion (Figure 1-(2)). This technique does not require further optimization as it precisely screens many crystallization conditions for one protein solution and one precipitant solution. Another advantage of thin capillaries is that a small amount of the protein solution is required for the experiment. Moreover, the protein crystals in capillaries can be also directly measured at the synchrotrons [10].  single capillary organisation that blocks fast diffusion of the protein and precipitant solutions by a physical barrier, typically formed by agarose or silica gel, known as gel acupuncture method (GAME) (Figure 1-(1, 3, 4)). Modifications (ii) and (iii) have been successfully used for the crystallization of not only globular proteins, but also RNA and DNA protein complexes and larger membrane protein complexes [10,11,12].

Crystallization in gels
Crystallization in gels is a suitable method for growth of high-quality macromolecular crystals. This method is effective due to the invariable environment enabling crystal growth, preventing the production of protein aggregates or sedimentation and slows diffusion of molecules in the crystallization chamber [13]. The most used gels for macromolecular crystallization are hydrophilic gels, namely agarose and silica gels, even though they have different properties triggering the crystal growth [14].
Regarding nucleation, agarose gels significantly promote it, whereas, silica gels inhibit this process, but the exact mechanism behind it remains unknown. The benefits of the use of agarose gels are (i) the concentration of agarose gel can be very low, (ii) agarose gel polymerises faster than silica gels, (iii) agarose gel can be employed in any crystallization technique, (iv) cofactors, ligands and even cryoprotectants are reported to be easily soaked into the crystal in the presence of the agarose gel, (v) agarose gels help to produce bigger crystals for neutron diffraction analysis and also (vi) serves as delivery medium for nanocrystals for X-ray freeelectron laser analysis (Figure 1-(3, 4)) [14][15][16][17].

Microgravity
The first attempts to confirm the substantial role of microgravity for crystal growth were done more than 35 years ago [18]. There was an assumption that absence of gravity can lead to growing of top quality crystals, thus crystallization experiments in the microgravity environment in the space shuttle were carried out. First experiments were performed by vapour diffusion method, later the counter diffusion and dialysis crystallization were applied as well [18,19]. The main advantage of growing protein crystals in space is that there is very diffusive mass transport and reduction of formation of possible crystal defects.
There are different views on the success of this method. In many cases, it was proven that space provides good conditions for growing better diffractive crystals against the results of crystals grown on Earth [20,21]. Many positive examples of growing the crystals in space show improved diffraction resolution, better diffraction intensity, reduction of crystal disordering and clustering, prolonged nucleation process, excluded crystal sedimentation and in some cases growing of larger crystals, sometimes also with different space groups [20,22]. Of course, the microgravity experiments are connected with some limitations that are mainly bureaucracy for flight approval of protein samples, delays of mission and with this connected possible degradation of protein or crystal samples [22].

Microfluidic chips
Recently, screening and crystallization by counter diffusion method has been improved by the application of microfluidic chips. Typically, the chips are composed of channels connected by a cross section. This organization of the crystallization chamber facilitates the screening of the wide range of the protein and precipitant concentrations; the supersaturation state is achieved by diffusion of the molecules [9]. Another advantage of this approach is the requirement of a small DOI: http://dx.doi.org/10.5772/intechopen.97162 amount of the protein and thus is suitable for proteins that are demanding to purify in larger volumes [23]. In practice, the protein sample is loaded into the eight channels at once by micropipettes and precipitant solutions are placed in the reservoirs [24]. The crystal growth in microfluidic chips is much faster than in other crystallization systems and the results are highly reproducible [25]. Microfluidic chips are also applicable for the microseeding strategy or soaking with the ligands [26]. The resulting crystals can be analysed by in situ serial crystallography [24,27].

Electric and magnetic field
The principle of the method benefiting from electric fields relies on the separation of the nucleation and crystal growth; it can be applied in the electric as well as the electromagnetic field. The nucleation process takes place while the current is switched off [28]. In order to perform crystallization experiments in the electric field, the specially designed crystal growth cell has to be used. The system contains two parallel electrodes and is connected to a direct current source that provides direct or alternating current. Nevertheless, the experiment has to be set up before the application of the current. It was reported that the current positively influences the size of protein crystals. Specifically, lower frequencies of alternating current provide less larger crystals compared to higher frequencies [28]. For the purposes of using electric current the e-crystallization cells based on vapour diffusion or batch method were made [29].
In the case of using magnetic fields, there is a similar outcome as carrying out an experiment in microgravity. This can be achieved when using a vertical magnetic field with use of anti-gravitational force. The crystals treated by magnetic fields show better diffraction quality as was revealed for microgravity experiments [30,31]. In addition, the crystallization conditions need to be known before initiating the experiment. Moreno 2017 mentions the use of gels for obtaining large well-arranged crystals together with use of magnetic forces for at best two days and more days. For this type of experiment, the capillary glass pipettes were reported to be suitable for use of homogeneous or nonhomogeneous magnetic fields [28]. The best influence of magnetic field use is regulation of quality and crystal size [32].

Seeding
The supersaturation level needed for the nucleation and the growth of best diffracting crystals is often different. Nucleation takes place when the supersaturation level is very high, while crystal-growing process appears when the conditions are slightly changing to lower supersaturation level [33]. Therefore, the seeding strategy has been developed as one of the most powerful tools for the optimization of the size and diffractive quality of single crystal. Another benefit is that it reduces the amount of the used protein and saves time needed for the spontaneous nucleation. This is also suitable for the examination of the slightly changing crystallization conditions, such as testing different pH ranges, adding new additives or even new precipitants. Several seeding strategies such as macroseeding, microseeding and/or their modifications can be applied.
In order to perform macroseeding, a single seed crystal has to be selected; its size is not relevant. The solubility of the crystal has to be investigated by testing of different precipitant concentrations to establish the conditions where the crystal is stable. This method is more demanding as multiple washes of the crystal are needed before the crystal is put into the suitable equilibration condition. The washes are aimed to eliminate additional nuclei. The main disadvantage of the method is that during this handling, the crystal seeds can be damaged and can form only the microcrystals in the new equilibration condition, which are not suitable for the X-ray diffraction analysis [33].
Microseeding can be described as a delivery of microseeds into a new equilibration condition. Before the experiment, the seed stock has to be prepared by crushing crystals using vortex, sonication or seed beads [34]. Once the seed stock is prepared, the dilution series have to be done in order to stabilize the nuclei presented in the seed stock and enable them to form suitable crystals in the new crystallization drop.
Another way of seeding strategy is the streak seeding. The drop containing the protein crystal is opened and the crystal is touched using an animal (cat, horse, rabbit or chinchilla) whisker. The nuclei can be attached to the whisker and transfer into a new crystallization drop. The sitting drop technique is recommended for this experiment, as it does not dry as quickly as the drops in hanging drop technique [33].
Cross seeding is specifically designed for crystallization of related proteins. These related proteins are different recombinantly modified protein variants, homologous proteins, and chemically modified proteins, macromolecular complexes with ligand or even misfolded proteins that are crystallizing difficult [35]. Some of the related proteins do not crystallize in the same conditions; however, sometimes this method can help and save the protein solution and time.
Random microseeding is a modified microseeding strategy using random screening kits. It is reported to create extra additional hits as well as produce better quality protein crystals for diffraction experiments [36].

Co-crystallization with ligands and crystallization chaperones
The co-crystallization of macromolecules with ligands have been developed because some proteins crystallize efficiently with ligands considering its structure stabilization role. The use of ligands has benefits not only for improving crystallization but also for obtaining stable and functional protein during purification because they enhance thermal stability of molecules. The study of small ligands stabilization roles for proteins in aqueous solution is still in progress. There exist online libraries of ligands as compounds used for crystallization purposes to prepare the best condition for protein stabilization [37]. The use of ligands for crystallization promotes the possibility of obtaining good-quality protein crystals. Ligands are regularly used to expand the screening capacity of commercially available screens [38]. On the contrary, it can be challenging to crystalize protein-ligand complex and so, the use of stabilizing additives is necessary. The process of co-crystallization can be further affected by temperature used for the complex formation prior its crystallization and by concentration of protein or used ligand [39].
Another "compounds" used for stabilization of proteins are amino acids and their derivatives such as glycine ethyl ester or glycine amide. There is noted considerable benefit of the stabilization role of amino acids for protein purification as well as crystallization [40,41]. Hence, small molecule additives such as coenzymes, prosthetic groups, inhibitors and small molecules, mentioned in [42], are used for successful crystallization of macromolecules. Usage of these additives helps forming hydrogen bonds and crosslinks between molecules that helps formation of stable crystals with regular lattice order of molecules [38].
Additionally, the antibodies and nanobodies are used to improve protein crystallization. Antibodies such as conventional IgG, Fab-fragments (fragment antigen binding), scFv (single-chain variable fragments) and Camelid hcAb (heavy chain IgG) were used to reinforce protein crystallization, mainly for membrane protein DOI: http://dx.doi.org /10.5772/intechopen.97162 co-crystallization. Latterly, nanobodies replaced the use of antibodies because production of antibodies is limited and obtained yield tends to be conformationally heterogeneous with limited solubility [43,44]. On the contrary, nanobodies are extremely stable and soluble antibody single-domain fragments that stabilize unstructured proteins by binding to them, form crystal contacts and thus speed up crystal growth. For crystallization of selected protein, many conformationally different nanobodies can be used to enhance the probability of best quality crystal hits [45].

Protein engineering
Protein engineering is one of the most used strategies in molecular biology to produce sufficient amounts of soluble protein. Protein biocrystallogenesis is likewise using protein engineering for crystallization of proteins that are hard to crystallize owing to have highly flexible regions, high surface entropy or less common high surface hydrophobicity or proteins are not able to be produced soluble [46]. To conquer these issues some strategies such as designing modified proteins, mutation of surface residues or designing fusion proteins are used [47].
The modifications of proteins include designing only some parts of protein such as conserved domains, and removing flexible parts or internal loops that prevent crystal formation. This can be fulfilled by creation of genetic constructs based on e.g. computation selection of expression clone libraries to find the clones with fragments that would encode the concerned part of the protein [47]. In the literature, several strategies for protein modification have been mentioned such as colony filtration blot technique [48], the open reading frame selection through the ESPRIT automation to find soluble complexes [49] and fluorescence screening based on protein fusion with GFP protein [47,50].
Next strategy to enhance crystal formation is design of protein mutants by sitespecific mutation or chemical modification. Mutagenesis is ordinarily used to produce soluble protein assuming that wild type is not soluble. The substitution of Lys, Gln or Glu that are frequently located on the molecule surface [51] with Ala that has lower surface entropy is a commonly used strategy [52]. There are plenty of protein structures that were solved mainly due to mutagenesis [47]. In addition, many chemical modifications as reductive methylation of Lys that reduce protein solubility and reductive carboxymethylation or substitution of Cys that on the contrary enhance solubility, stability and have benefits for protein function and oligomerization [52]. Different strategy is to introduce Cys at the surface to strengthen formation of symmetrical dimers that seem to crystallize more easily [52,53].
Design of fusion proteins or fusion tags is another approach to produce soluble proteins and subsequently solve protein structures. In many cases the fusion partner such as short Strep-tag or poly-histidine tag and some larger tags as glutathione S-transferase (GST) and Maltose-binding protein (MBP) are removed before crystallization [54][55][56]. This is done to remove possible highly flexible regions that can block formation of protein contacts during crystallization. However, it was shown that in two different approaches fusion proteins manage to crystalize target proteins. First, one is based on increasing protein surface area by incorporation of fused protein such as MBP, Green fluorescent protein (GFP), Barnase or T4 lysozyme (T4L) [56]. Second is aimed to merge interacting proteins or protein and peptide with linker, mostly to stabilize one of fused proteins or to support their interaction [56,57].

Crystallization in living cells
The formation of native protein crystals in living cells has been detected over the last few decades as a natural process. This approach is ideal for proteins that cannot be crystallized applying basic crystallization techniques. Recently, X-ray sources have been exceedingly improved, such as microfocus beamlines, X-ray free-electron lasers, or even serial crystallography as a new data collection strategy [58,59]. These advances in the field of X-ray crystallography enable data collection from smaller crystals and thus enable the development of in cellulo crystallization as a powerful advanced crystallization approach.
Recombinant or fusion recombinant proteins can be crystallized in a number of living cells, including plant cells, mammalian cells or insect cells [60]. The bottleneck of this strategy is the detection of the protein crystals inside the cells. Crystals can occur in different compartments of the cell, such as cytosol, endoplasmic reticulum (ER) or peroxisomes [60,61]. The crystals can be detected using various methods, e.g. bright-field microscopy techniques or transmission electron microscopy (TEM), which is also used for the observation of crystal growth and subsequent optimization. The crystal can be isolated from the cell before the diffraction experiment or directly measured in the cell [60,62].

Automation
The expansion of the crystallographic field and running extensive research led to need for facilitation of performing crystallization experiments. This was achieved by automation that supported faster progress of experiments thanks to development of a variety of robots [63]. There are robots adjusted to establishment of crystallization conditions, arrangement of drops, for carrying out the vapour diffusion experiments specifically hanging drop, microbatch, free-interface diffusion and random micro-seeding seeding methods with sitting drop method as the most preferred method. Most widely used crystallization robots are from the Oryx series (Douglas Instruments Ltd.), the Mosquito (TTP Labtech, Royston, UK) and TOPAZ system (Fluidigm Corp., San Francisco CA, USA) [64]. Automation devices allow crystallization of nanovolumes into an array of plates for 96 or more conditions [28]. Each robot has its own specification, from which can be chosen preferred experimental method suitable for protein of interest.

Conclusion
In conclusion, a growth the good-quality macromolecular crystal is crucial for the determination of its atomic structure using of X-ray crystallography. Several effective advanced methods leading to the formation of crystal in diffraction quality are available. Nowadays, the extensive improvement of X-ray facilities enables data collection from smaller crystals and allows using the new trends in crystallization strategies that are developing as a powerful advanced crystallization approach.
The protein crystallization manufacturers such as Hampton Research, Molecular Dimensions Ltd., PerkinElmer, GE Healthcare, Danaher, Bruker, Agilent, Jena Bioscience, Rigaku, Formulatrix and MiTeGen are basic companies responsible for systematic invention of new products and technologies that constantly improve protein crystallization methods and are looking for crystallization techniques for hardly crystallisable proteins such as membrane proteins.
Next essentials that have an effort to improve crystallization techniques are attempts to use crystals in different non-conventional ways. One of them is sending biosubstances (protein-based medicines) into space to try to crystallize them for finding new uses of crystallized proteins as pharmacotherapeutics. These protein crystals can be supposedly thereafter used for long-term storage or for stabilization of biosubstances by transferring them into solid state (Bristol-Myers Squibb Company). Another use of crystallization was reported in material sciences, more precisely for improvement of electronic materials developed on the base of biomolecules arrangement in crystals [65].
These and many more applications of protein crystallization are reasons for development of new methods and strategies for faster crystallization and obtaining of well diffractive crystals and for reduction of protein volume. However, there is still a necessity for improvement and new methods and strategies for biomolecules crystallization awaits to be discovered. Although this can be achieved more easily because biochemistry, biotechnology, physics, chemistry and applied nanosciences are extra overlapped and allow to improve and explain in depth the process of protein crystallization.