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The self-assembly of biomacromolecules is an extremely important process. It is potentially useful in the fields of life science and materials science. To carry out the study on the self-assembly of proteins, it is necessary to find out the suitable self-assembly conditions, which have always been a challenging task in practice. Inspired by the screening technique in the field of protein crystallization, we proposed using the same screening technique for seeking suitable protein self-assembly conditions. Based on this consideration, we selected 5 proteins (β-lactoglobulin, hemoglobin, pepsin, lysozyme, α-chymotrypsinogen (II) A) together with 5 screening kits (IndexTM, BML, Morpheus, JCSG, PEG/Ion ScreenTM) to investigate the performance of these crystallization screening techniques in order to discover new optimized conditions of protein self-assembly. The screens were all kept at 293 K for certain days, and were analyzed using optical microscope, scanning electron microscope, transmission electron microscope, atomic force microscope, fluorescence microscope, and atomic absorption spectroscope. The results demonstrated that the method of protein crystallization screening can be successfully applied in the screening of self-assembly conditions. This method is fast, high throughput, and easily implemented in an automated system, with a low protein consumption feature. These results suggested that such strategy can be applied to finding new conditions or forms in routine research of protein self-assembly. KEY POINTS: • Protein crystallization screening method is successfully applied in the screening of self-assembly conditions. • This screening method can be applied on various kinds of proteins and possess a feature of low protein consumption. • This screening method is fast, high throughput, and easily implemented in an automated system.

The joining of amorphous alloys and common metals has been hot subjects of abundant studies in recent years. Nevertheless, the crystallization and fracture of amorphous alloy are still big challeng...

In recent years, with the rapidly increasing demand for pure protein products in various fields (biomedicines, biochemical reagents, food industries, etc.), the need for low-cost, high-quality prot...

Protein crystallization is a process that is very sensitive to the physical environment. Audible sound is an environmental characteristic that can significantly affect the crystallization process. Previously, it was found that the crystallization result is frequency dependent, that is, the crystallization of protein under different sound frequencies yields different results. Here, we further investigate the effect of varying frequency (or a frequency program) on protein crystallization. Twelve different frequency programs and six proteins were used to test the effect of varying sound frequency on protein crystallization. The results showed that varying the audible sound frequency from high to low exhibited the most significant improvement in protein crystallization. Varying frequency linearly from 15 000 to 100 Hz in 12 h best promoted crystallization, with the average number of crystallization hits 36.5% higher than in the control. Crystal quality was improved with sound irradiation using STW2 program. O...

The immobilization of iodine waste is a challenge in the field of nuclear waste. In this work, iodine waste (silver-coated silica gel with different iodine loadings) was immobilized at low-sintering temperatures (500, 550, and 600 ​°C). XRD represented that the crystallized phases of the sintered matrix consist of AgI and B2O3. The crystallization behavior of boron can be found as the sintering temperature increased from 500 ​°C to 600 ​°C. FT-IR results proved that in the process of crystallization, part of the microstructure related to B–O stretching transformed from [BO4] to [BO3] units, which benefits low-temperature iodine waste immobilization due to the bonding energy of [BO3] is higher than that of [BO4]. Our work reveals the B-related microstructure transition in low-temperature immobilization iodine waste.

Protein crystallization is of great importance because protein crystals have a number of different important applications, including large-scale purification of proteins, determination of protein structure, nanoparticle preparation, and theoretical studies of crystallization. An approach often used to efficiently crystallize proteins is the use of nucleants or seeds (small fragments of protein crystals) that can help increase the probability of protein crystallization. Due to the very positive effect that seeding has on protein crystallization, seeds are now widely accepted and utilized in practical protein crystallization. Here, we show that cross-linked protein crystals (CLPCs), which retain the crystal structure but are much more stable than non-cross-linked crystals, can also be used as a new type of seed for promoting protein crystallization. Seeding with CLPCs has effects on both the reproducibility and screening of protein crystals and could improve the optical perfection (well-defined facets) of p...

Protein crystallization screens are commonly used to find specific crystallization conditions. In practice, undesirable clear drops are often ignored. We hypothesize that clear drops may be potentially useful for finding the conditions leading to proteins with high solubility and monodispersity . Based on this idea, we first tested three model proteins and confirmed that improved monodispersity could be observed in the clear drops. Next, we used this strategy to achieve the maximum solubility and monodispersity of a new protein (RNA binding motif 3, RBM3). We suggest that this strategy is useful not only for high-throughput screening of optimal solubilization conditions but also for screening of biopharmaceutical formulations.

Finding new nucleants to promote protein crystallization is an important task, especially for purposes other than structural determination. Here, we investigated cyclodextrins and its derivative particles, as potential nucleants for protein crystallization. β-cyclodextrin (β-CD) and its derivatives (including p-toluenesulfonyl-β-cyclodextrin (PTCD), polymer-β-cyclodextrin (PCD), Mono-(6-(1,6-hexamethylenediamine)-6-deoxy)-β-Cyclodextrin (MHCD) and Mercapto-β-cyclodextrin (MCD)) were used as nucleants. The experimental results confirmed that β-CD and its derivatives showed significantly positive effects, promoting protein crystallization and improving crystal quality. A larger number of protein molecules (including lysozyme, catalase, subtilisin A VIII, concanavalin A VI, α-chymotrypsinogen, proteinase K, cellulase, papain, glucose isomerase, hemoglobin and ribonuclease A XII) attached to the particles usually corresponded to a higher crystallization success rate. More detailed analysis showed that cyclode...

We have proposed a rational strategy for selecting a suitable pH of protein solution based on protein biochemical properties. However, it is difficult to use this strategy for biochemical properties unknown proteins. In this paper, a simpler and faster pH buffer strategy was proposed. An additional pH-controlling buffer was added to crystallization droplet mixed with protein solution and commercial crystallization reagents to adjust its pH. The results revealed that protein crystallization success rates were enhanced by this strategy due to expansion of the pH screening space, which was closely related with protein solubility. Thus, the possibility of reaching supersaturation was increased by using this strategy.

The elucidation of protein structures by X-ray crystallography remains the most effectual method to provide accurate structural details at atomic resolution for rational drug design and other biotechnological research studies. Also, emerging applications of protein crystals as ordered nanostructure scaffolds for catalysis, imaging, and drug delivery are attracting much attention. However, the first step of these applications is obtaining high-quality crystals, which is still an obstacle. Successful crystallization requires two steps: nucleation and crystal growth, while the nucleation is a precondition for harvesting the crystal of interest. So controlling protein nucleation may be an alternative breakthrough for this bottleneck. It is well known that nucleants can induce protein crystallization and improve crystal quality, so investigation on the nucleants that can be universally used for any protein crystallization is ongoing. This manuscript reviews the advances that have been achieved using nucleants in protein crystallization and it is a suitable reference for practical crystallization.

Bio-based unsaturated poly(butylene adipate-co-butylene itaconate) (PBABI) aliphatic copolyesters were synthesized with pentaerythritol (PE) as a modifier, observing the melting point, crystallization, and glass transition temperatures were decreased from 59.5 to 19.5 &deg, C and 28.2 to &minus, 9.1 &deg, C as an increase of itaconate concentration, and Tg ranged from &minus, 54.6 to &minus, 48.1 &deg, C. PBABI copolyesters tend to the amorphous state by the existence of the BI unit above 40 mol%. The yield strength, elongation, and Young&rsquo, s modulus at different BA/BI ratios were valued in a range of 13.2&ndash, 13.8 MPa, 575.2&ndash, 838.5%, and 65.1&ndash, 83.8 MPa, respectively. Shear-thinning behavior was obtained in all BA/BI ratios of PBABI copolyesters around an angular frequency range of 20&ndash, 30 rad s&minus, 1. Furthermore, the thermal and mechanical properties of PBABI copolyesters can be well regulated via controlling the itaconic acid contents and adding the modifier. PBABI copolyesters can be coated on a 3D air mesh polyester fabric to reinforce the mechanical property for replacing traditional plaster applications.

Protein crystallizes at specific conditions (correct precipitants, appropriate concentrations of protein and precipitants, suitable pH and correct temperature, etc.). If the conditions are not appropriate, crystallization will not occur. In protein crystallization screening, the target protein is mixed one by one with many chemical agents and then incubated at a set temperature. If the concentrations of the chemical agents and the target proteins are not in a range suitable for crystallization, the crystallization will not occur. To expand the concentration screening space, we propose in this paper a new design of protein crystallization plates for cross-diffusion microbatch and microbatch methods. The new plates have 96 units corresponding to the conditions of the commercial screening kits, and each unit contains four wells for holding the crystallization droplets. By dispensing crystallization droplets to the four wells at different volume ratios of protein to precipitant solutions, we can obtain four d...

The successful crystallization of proteins is important because their molecular three-dimensional structures can be obtained through X-ray diffraction when in their crystalline form. Investigation of the crystallization process is beneficial for this purpose. We have reported that protein crystallization is sensitive to audible sound, which is commonly present but is often ignored. Here we investigate the effect of audible sound parameters, especially frequency, on a protein crystallization. We show a significant facilitation of protein crystallization using 5000 Hz audible sound, possible mechanism was also tried to be clarified. Suitably controlled audible sound can be beneficial for promoting protein crystallization. Therefore, audible sound can be used as a simple tool to promote protein crystallization. In addition, the processing of other types of materials may also be affected by audible sound.

Environments with varying temperatures have been shown to beneficially increase the probability of obtaining protein crystals. Therefore, a cycling temperature strategy (CTS) has been proposed for protein crystallization screening. During the practical application of this strategy, it is necessary to know the effective temperature range that promotes crystallization to design a suitable temperature program. In this paper, the effects of different temperature ranges on lysozyme crystallization (or more specifically, nucleation) were investigated. The results show that a small periodic variation in the temperature range of as little as 0.4 K can have a significant effect on the crystallization success rate under some crystallization concentration conditions, confirming that crystallization of lysozyme is very sensitive to temperature variation. Because practical protein crystallization is always performed in an environment with slight temperature variations, the sensitivity of protein crystallization to temperature may provide an explanation for the poor reproducibility of protein crystallization. Further investigation of the CTS on lysozyme crystallization showed that a cycling temperature strategy exerts an effect on protein crystallization by altering the supersaturation caused by changes in temperature.

A facile approach was developed to prepare highly dispersed TiO2 nanoparticles with selected phase. The crystallization phase of the nanoparticles can be easily tuned from anatase to rutile by the dosage of hydrochloric acid in the reaction system. The crystallite size of the as-prepared anatase TiO2 nanoparticles was ca. 3.2 nm with high dispersion. A transparent TiO2 colloid was obtained by dispersing the as-prepared anatase TiO2 nanoparticles in deionized water without any organic additives added. The concentration of TiO2-H2O colloid can be as high as 1600 g/L. The optical transmittance of TiO2-H2O colloid with a low concentration was nearly 100% in the visible region. Furthermore, anatase TiO2 nanoparticles(TiO2-NPs) showed superior photocatalytic performance compared to rutile TiO2-NPs.

Protein drugs are attracting increasing attention. Eight of best-selling drugs in worldwide are proteins. Crystallization is an important protein purification method; however, it remains a bottleneck step. Heterogeneous nucleate is an effective method of enhancing protein crystallization. Cyclodextrin has been widely used in pharmaceutics, and our previous study showed that it is an effective heterogeneous nucleate for enhancing protein crystallization, and it may be widely used in purification of protein drugs. However, cyclodextrin must be added into the crystallization plate wells by hand, which is labor intensive and is not beneficial to automated crystallization screening. We grafted β-cyclodextrin (β-CD) and its derivatives (i.e., p-toluenesulfonyl-β-cyclodextrin [PTCD], mono-(6-(1,6-hexamethylenediamine)-6-deoxy)-β-cyclodextrin [MHCD] and mercapto-β-cyclodextrin [MCD]) on crystallization plates and tested their effects on protein crystallization. Protein crystallization success rate was improved, particularly for the PTCD-grafted group. This is an easy method to facilitate protein crystallization and can be widely applied for automatic crystallization screening.

Protein crystallization is sensitive to the environment, while audible sound, as a physical and environmental factor during the entire process, is always ignored. We have previously reported that protein crystallization can be affected by a computer-generated monotonous sound with fixed frequency and amplitude. However, real-world sounds are not so simple but are complicated by parameters (frequency, amplitude, timbre, etc.) that vary over time. In this work, from three sound categories (music, speech, and environmental sound), we selected 26 different sounds and evaluated their effects on protein crystallization. The correlation between the sound parameters and the crystallization success rate was studied mathematically. The results showed that the real-world sounds, similar to the artificial monotonous sounds, could not only affect protein crystallization, but also improve crystal quality. Crystallization was dependent not only on the frequency, amplitude, volume, irradiation time, and overall energy of the sounds but also on their spectral characteristics. Based on these results, we suggest that intentionally applying environmental sound may be a simple and useful tool to promote protein crystallization.

One well-known prerequisite for successful crystallization from solution is a supersaturated solution. To achieve supersaturation, many methods are known, among which solvent evaporation is a common approach. For protein crystallization, the most widely used method is vapor diffusion, in which solvent evaporation from the crystallization solution is the major reason for achieving supersaturation. The solvent evaporation rate may affect the actual concentration distribution in the crystallization solution, thereby influencing the crystallization process. To explore the effect of evaporation rate on protein crystallization, we used lysozyme as a model protein and studied the crystallization success rate at different evaporation conditions. Successful crystallization occurred only when both supersaturation and evaporation rates were in suitable ranges. This study demonstrates that both supersaturation level and the rate of reaching supersaturation (or solvent evaporation rate) are important for lysozyme crystallization. To increase the chance of obtaining crystals, manipulation of solvent evaporation rate is one choice. According to this assumption, we performed crystallization screening trials at different evaporation rates using three model proteins. The trials demonstrate that control of the evaporation rate during crystallization may provide more opportunities to obtain crystals.

Crystallisation of proteins is usually achieved with the help of chemical agents. Because there are few general guidelines in determining what agents will help to crystallise a specific protein, suitable crystallisation agents are often found via exhaustive trial-and-error tests by mixing many chemical agents (the collection of which is called a crystallisation screening kit) one-by-one with the protein. Currently, many commercially available crystallisation screening kits have been developed and utilised in practical crystallisation screen experiments. However, information regarding the design of new screening kits has yet to be expanded using a large amount of experimental data. Here, we show the step-by-step design processes of a polyethylene glycol-based screening kit. It was found that the screening performance could be improved by modifying the crystallisation screening kits according to the accumulated data (such as those in the Biological Macromolecule Crystallisation Database (BMCD)), the screening test results and existing knowledge. The screening kit designed in this paper can be used for practical protein crystallisation screen experiments and the method can be used in the design of other crystallisation screening kits.

We present a new method for the realization of high-throughput protein crystallization screening using an array of 96 capillaries aligned in a circle. In this method, each capillary represents a single crystallization condition, and all capillaries experience identical magnetic field conditions. After crystallization, the crystals in the capillary can be directly diffracted without harvesting. This method proved easy to perform and is applicable for use in magnetic fields and may be further extended for use in other circumstances, for example, under space microgravity conditions.

This report describes the modification of crystallization plates by simply oxidizing the surface of the protein wells. The oxidized crystallization plates were tested in standard protein crystallization screening and reproducibility studies. The results showed that the protein wells of the treated plates were smoother and more optically transparent than those of the untreated plates, and more importantly, protein crystallization was significantly promoted after the oxidation treatment. Because there is no change to the routine screening protocol, this method is simple and easy to apply in protein crystallization.

Cross-linked protein crystals (CLPCs) are very useful materials in applications such as biosensors, catalysis, and X-ray crystallography. Hence, preparation of CLPCs is an important research direction. During the preparation of CLPCs, an often encountered problem is that cracks may appear in the crystals, which may finally lead to shattering of the crystals into small pieces and cause problem in practical applications. To avoid cross-link induced cracking, it is necessary to study the cracking phenomenon in the preparation process. In this paper, we present an investigation on how to avoid cracking during preparation of CLPCs. An orthogonal experiment was designed to study the phenomenon of cross-link induced cracking of hen-egg white lysozyme (HEWL) crystals against five parameters (temperature, solution pH, crystal growth time, glutaraldehyde concentration, and cross-linking time). The experimental results showed that, the solution pH and crystal growth time can significantly affect cross-link induced cracking. The possible mechanism was studied, and optimized conditions for obtaining crack-free CLPCs were obtained and experimentally verified.

The pH of a solution is an important parameter in crystallization that needs to be controlled in order to ensure success. The actual pH of the crystallization droplet is determined by the combined contribution of the buffers in the screening and protein solutions, although the contribution of the latter to the pH is often ignored. In this study, the effects of the buffer and protein solution pH values on the results of screening are systematically investigated. It was found that these parameters significantly affected the results and thus the following strategy for the selection of appropriate pH values is proposed: (i) when screening with only one protein solution, the pH should be as low, as high or as divergent from the pI as possible for a basic, acidic or neutral protein, respectively, within its stable pH range; (ii) when screening with two protein solutions, the pH values should be well separated from one another; and (iii) when multiple pH values are utilized, an even distribution of pH values is the best approach to increase the success rate of crystallization.

Nanocomposites of poly (vinyl alcohol) and antimony-doped tin oxide (ATO) were prepared by solution blending. The PVA composites were characterized by FTIR, TGA, DSC, WXRD and stress-strain testing. It has been found that adding ATO to the matrix has great influence on the crystallization behavior and glass transition temperature of PVA. The mechanical properties of PVA changed with the filler content, exhibiting an initial increase in these properties due to polymer-filler interactions. After a maximum value, at about 5 wt%, the mechanical properties decreased. Thermal stability of the nanocomposites was found to be remarkably enhanced by the incorporation of ATO.

Varying the temperature has been proven to be beneficial for improving the screening efficiency of protein crystallisation, and thus a crystallisation screening strategy based on this phenomenon can be developed. Such a temperature varying strategy can be applied in practical crystallisation screening, however, there are no guidelines for determining what temperature programme should be utilised. It is therefore necessary to investigate how the temperature programme affects the crystallisation process, so as to help people design a suitable temperature programme. For this purpose, we investigated the effect of temperature programmes on the protein crystallisation (lysozyme, proteinase K, and concanavalin A) that are characterised by different solubility behaviours with respect to temperature. Judging from the reproducibility studies of protein crystallisation with different temperature programmes, we recommend using linear temperature programmes for a moderate time period (24 to 48 h) and a large temperature range according to the properties of the proteins. (© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Temperature is generally considered as an important factor in protein crystallization. Such is true because crystals usually grow at a preferable temperature in a certain crystallization solution. If a nonsuitable temperature is used, the solution will not yield crystals. However, it is difficult to decide the best temperature suited for screening the crystallization condition of proteins. In this study, it was found out that, compared to constant temperature, a variation in a reasonable range can result in a more efficient crystallization screening. Using the Sparse Matrix Screen with the screening kit Index, this study tested nine commercially available proteins and proved that, compared to the conventional constant temperature strategy, a varying temperature strategy can actually increase the possibility of obtaining crystals. Consequently, the cycling temperature strategy (CTS) is then proposed to be utilized in most screening tasks when the suitable crystallization temperature is unknown.

Polymer blends consisting of linear poly(phenylene sulfide) (PPS) and hyperbranched PPS (HPPS) were obtained in melt. The solid-state properties of PPS and their blends were investigated by scanning electron microscopy (SEM), thermogravimetric analyzer (TGA), extraction measurement, differential scanning calorimetry (DSC) and dynamical mechanical analysis (DMA). Blends prepared by melt mixing turned out to be reactive as shown by the TGA and extraction measurement. SEM indicated that no phase separation occurs in PPS/HPPS blends. The degree of crystallization of the blends decreased with increasing HPPS content. Both the storage modulus and loss modulus increased as HPPS content increasing.

Protein crystallization is affected by many parameters, among which certain parameters have not been well controlled. The temperature at which the protein and precipitant solutions are mixed (i.e., the ambient temperature during mixing) is such a parameter that is typically not well controlled and is often ignored. In this paper, we show that this temperature can influence protein crystallization. The experimental results showed that both higher and lower mixing temperatures can enhance the success of crystallization, which follows a parabolic curve with an increasing ambient temperature. This work illustrates that the crystallization solution preparation temperature is also an important parameter for protein crystallization. Uncontrolled or poorly controlled room temperature may yield poor reproducibility in protein crystallization.

High-quality protein crystals of suitable size are an important prerequisite for applying X-ray crystallography to determine the 3-dimensional structure of proteins. However, it is often difficult to obtain protein crystals of appropriate size and quality because nucleation and growth processes can be unsuccessful. Here, we show that by adsorbing proteins onto porous polystyrene-divinylbenzene microspheres (SDB) floating on the surface of the crystallisation solution, a localised high supersaturation region at the surface of the microspheres and a low supersaturation region below the microspheres can coexist in a single solution. The crystals will easily nucleate in the region of high supersaturation, but when they grow to a certain size, they will sediment to the region of low supersaturation and continue to grow. In this way, the probability of crystallisation and crystal quality can be simultaneously increased in a single solution without changing other crystallisation parameters.

Blast furnace (BF) slag in south-western part of China contains large amount of titanium. For the purpose of beneficial utilization of titanium in the slag, microwave (MW) processing of the slag has been proposed. In this study, a fundamental research on heating behavior of the slag by application of 2.45 GHz MW was performed.The slag specimens can be heated in a domestic MW oven at constant input power (0.5 kW) and sudden increase of the temperature is observed after several heating cycles. A phenomenon of so-called thermal runaway (TRW) occurred. A part of the specimen melted, and the XRD analysis indicated that this area became amorphous. Crystallization of the CaTiO3 phase occurred by MW heating of the melted slag at 600°C.A sintered CaTiO3 compact was heated by MW much more than the synthetic Chinese slag. The synthetic slag without containing Ti was not heated very well. Permittivity of CaTiO3 (loss factor) was measured and demonstrated to be much larger than the other oxides in the slag. Therefore, it is concluded that CaTiO3 phase is responsible for heating of the whole slag.

Protein crystallization under a magnetic field is an interesting research topic because a magnetic field may provide a special environment to acquire improved quality protein crystals. Because high-quality protein crystals are very useful in high-resolution structure determination using diffraction techniques (X-ray, neutron, and electron diffraction), research using magnetic fields in protein crystallization has attracted substantial interest; some studies have been performed in the past two decades. In this research field, the hardware is especially essential for successful studies because the environment is special and the design and utilization of the research apparatus in such an environment requires special considerations related to the magnetic field. This paper reviews the hardware for protein crystallization (including the magnet systems and the apparatus designed for use in a magnetic field) and progress in this area. Future prospects in this field will also be discussed.

High-quality crystals are key to obtaining accurate three-dimensional structures of proteins using X-ray diffraction techniques. However, obtaining such protein crystals is often a challenge. Several containerless crystallization techniques have been reported to have the ability to improve crystal quality, but it is unknown which is the most favourable way to grow high-quality protein crystals. In this paper, a quality comparison of protein crystals which were grown under three containerless conditions provided by diamagnetic levitation, silicone oil and agarose gel was conducted. A control experiment on a vessel wall was also simultaneously carried out. Seven different proteins were crystallized under the four conditions, and the crystal quality was assessed in terms of the resolution limit, the mosaicity and theRmerge. It was found that the crystals grown under the three containerless conditions demonstrated better morphology than those of the control. X-ray diffraction data indicated that the quality of the crystals grown under the three containerless conditions was better than that of the control. Of the three containerless crystallization techniques, the diamagnetic levitation technique exhibited the best performance in enhancing crystal quality. This paper is to our knowledge the first report of improvement of crystal quality using a diamagnetic levitation technique. Crystals obtained from agarose gel demonstrated the second best improvement in crystal quality. The study indicated that the diamagnetic levitation technique is indeed a favourable method for growing high-quality protein crystals, and its utilization is thus potentially useful in practical efforts to obtain well diffracting protein crystals.

The nucleation zone has to be reached for any crystal to grow, and the search for crystallization conditions of new proteins is a trial and error process. Here a convenient screening strategy is studied in detail that varies the volume ratio of protein sample to the reservoir solution in the drop to initiate crystallization that is named “composition modification”. It is applied after the first screen and has been studied with twelve proteins. Statistical analysis shows a significant improvement in screening using this strategy. The average improvement of “hits” at different temperatures is between 32 and 42%, for examples, 41.8% ± 14.0% and 35.7% ± 12.4% (± standard deviation) at 288 K and 300 K, respectively. Remarkably, some new crystals were found by composition modification which increased the probability of reaching the nucleation zone to initiate crystallization. This was confirmed by a phase diagram study. It is also demonstrated that composition modification can further increase crystallisation success significantly (1.3 times) after the improvement of “hits” by temperature screening. The trajectories of different composition modifications during vapour diffusion were plotted, further demonstrating that protein crystallizability can be increased by hitting more parts of the nucleation zone. It was also found to facilitate the finding of initial crystals for proteins of low solubility. These proteins gradually become more concentrated during the vapour diffusion process starting from a larger protein solution ratio in the initial mixture.