**2. Advanced biocrystallogenesis methods**

#### **2.1 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.

#### **2.2 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].

*Advanced Biocrystallogenesis DOI: http://dx.doi.org/10.5772/intechopen.97162*

#### **Figure 1.**

*Advanced biocrystallogenesis methods. 1. Free-interface diffusion, 2. Crystallization in capillaries, 3. Counterdiffusion in capillaries, 4. Crystallization in gels and GAME (prepared by author Barbora Kascakova).*

Three different organizations of capillaries can be applied for counter-diffusion experiments: (i) simple one chamber organization composed of the protein and precipitant solutions, (ii) two chamber single capillary organization is formed by the precipitant solution and the protein solution mixed with the gel and (iii) three chamber 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 [10–12].

#### **2.3 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)) [14–17].

#### **2.4 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].

#### **2.5 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

#### *Advanced Biocrystallogenesis 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].
