Gold nanoparticles induce protein crystallization

Nucleation of protein crystals by gold nanoparticles was observed. Lysozyme and ferritin were used as model proteins. The effect was established with uncoated gold nanoparticles and with gold nanoparticles coated by 16‐mercaptodecanoic acid. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Nature of impurities during protein crystallization

Crystallography Reports - Lysozyme crystal growth was studied using reagents of different purity of three trademarks— Seikagaku Corporation (sixfold recrystallized lysozyme), Sigma-Aldrich...     

Simplex optimization of protein crystallization conditions

Simplex algorithms have been used to optimize for size, number and morphology of lysozyme and apoferritin crystals. This approach requires fewer experiments than the single-factor-at-a-time method or factorial designs and will be useful in conserving materials on the International Space Station. The simplex method has the possible advantage that it conserves on materials by reducing the number of experiments required to optimize a crystallization system. The process is iterative and exploratory and should allow optimum microgravity conditions to be determined which might very well be different from the optimum conditions on Earth. Because the simplex method uses simple mathematical operations to calculate the next set of crystallization conditions it will be easier for crystal growers to implement than factorial designs. Factorial experiments are based on varying all factors simultaneously at a limited number of factor levels. This results in a model that is used to determine the influence of each factor and their interactions. Factorial design experiments are especially useful at the beginning of an experimental study and as a screening tool to investigate a large number of factors. The simplex method is an optimization method which is model-independent and requires no fitting of models to data. Also, when applied to protein crystal growth the simplex method does not rely on an absolute quality score. Instead, with each iteration a comparison is made to the last experiment and the results are assigned as being “better or worse”. In this study, commercially obtained apoferritin was purified from 65% monomeric apoferritin to 92% monomeric apoferritin by size exclusion chromatography. Simplex optimization found the best apoferritin crystals were obtained at 15 mg/ml apoferritin, 2.0% CdSO₄, 25°C using the hanging drop vapor diffusion method of crystallization and at 24 mg/ml apoferritin, 1.5% CdSO₄, 25°C using the containerless crystallization method. For lysozyme, the simplex method found the best crystals at 19 mg/ml lysozyme, 7.0% (w/v) NaCl, pH 4.0, 25°C using the hanging drop vapor diffusion method of crystallization. For both proteins, the optimum conditions were found with less than ten experiments using very little protein. Finally, we report that the factors to be considered in the successful application of this method to crystallization are the number of variables to be studied, the initial conditions, step size and analysis of crystal quality.     

The effect of diluting crystallization droplets on protein crystallization in vapor diffusion method

In this study, effects of diluting either protein or crystallization agents in the droplets on the success rate of protein crystallization was investigated. Diluting the crystallization agent was found to increase the success rate of protein crystallization. Theoretical analysis showed that, concentration ranges of both protein and crystallization agent that can be scanned during the vapor diffusion process are wider with diluting the crystallization agent than that without dilution, resulting in more opportunities for the crystallization solution to be in the nucleation zone. On the other hand, diluting protein could lead to controversial results depending on the location of the initial concentration relative to that of the nucleation zone in the phase diagram. The method of diluting the crystallization agent is therefore proposed as an alternative modification to the conventional vapor diffusion method for obtaining more crystallization conditions in protein crystallization screening. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Bond selection during protein crystallization: Crystal shapes

The so‐called bond selection mechanism, BSM (C.N. Nanev, Progress in Crystal Growth and Characterization of Materials, 59 , 133–169, 2013) allows explaining a set of traits in both protein crystal nucleation and growth processes. BSM explanatory and predictive power are enhanced now, when intra‐crystalline repulsive interactions are assumed to act in parallel with the attractive forces, the former arising due to protein surface patch‐to‐patch incompatibility. Shapes of 1D and 2D protein crystals are considered from such a perspective. Using BSM the strong directional kinetic anisotropy in the edge growth rates of 2D protein crystals is tackled. The shapes of near‐critically sized apoferritin crystals and of experimentally grown 3D apoferritin crystals are considered.     

The factors during protein crystallization: A review

Many factors have effects on crystallization. The major influencing factors during protein crystallization are summarized in this paper. Recommendations are made about the basic process of crystallization and the analysis of the thermodynamic effects on crystallization, and the dynamics factors effecting crystallization during an equilibrium process are discussed. The advantages of ionic liquid in various chemical processes, especially in the field of biochemistry, are emphasized, and some tentative conclusions are made about future short-term trends. The text was submitted by the authors in English.     

Estimation of the effect of relative humidity on protein crystallization

The effect of relative humidity in a crystallization box on the rate of establishment of supersaturation conditions during protein crystallization by diffusion of solvent vapors is estimated. A modified crystallization box is designed, which provides the formation of a stable air flow with a specified relative humidity and its measurement directly in the closed space between a drop and a reservoir. The range of relative humidities necessary to obtain the supersaturation conditions in a drop with a protein crystallization solution is determined.     

Sedimentation as a tool for crystallization from protein mixtures

Crystals from apoferritin which is an iron‐free form of protein ferritin were obtained from protein mixtures lysozyme/apoferritin using sedimentation under high gravity. Solution containing apoferritin at concentration as high as 5mg/ml in the presence of 25mg/ml lysozyme and overlaid on 5%(w/v) CdSO₄ in 0,2M/L NaAC, pH=5 still favors apoferritin crystal formation under normal gravity conditions, but at apoferritin concentrations <0,5mg/ml (∼1,14µM/L) in 25mg/ml (∼1,71mM/L) lysozyme only the sedimentation in a centrifuge appears to be useful for separating the apoferritin molecules from the mixture followed by apoferritin crystallization in the same system. The very high molecule number ratio (∼1:10³) of two proteins is used to stress on the observed effect. (© 2006 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Understanding protein crystallization on the basis of the phase diagram

The phase diagram of a protein–water system is described with a simple model with parameters for the interaction between the protein molecules in the crystal and in the solution. For a certain range of these parameters the phase diagram shows a metastable liquid–liquid immiscibility region. It is shown that this region corresponds to the “crystallization slot” for growing crystals, as proposed by George and Wilson [Acta Crystallogr. D 50 (1994) 361]. Nucleation in this region proceeds in two steps. First small liquid droplets with a high protein concentration are formed; then small crystalline nuclei grow inside these droplets. In the crystallization slot crystals are covered by a thin liquid film with a high protein concentration. We discuss NMR experiments on lysozyme, which show that nucleation is a transient process with an induction time.     

Mechanism of aggregation and intergrowth of crystals during bulk crystallization from solutions

Available literature data on aggregation kinetics of crystals of a number of salts during their bulk crystallization from solutions have been analysed. The proposed earlier mechanism of aggregation and intergrowth of crystals during bulk crystallization owing to formation of nucleus‐bridges between crystals was tested and confirmed. The aggregation kinetics of crystals was described by the familiar Smoluchowski equation for coagulation of colloidal particles. However, in a bulk crystallization process, the aggregation constant in this equation decreased as supersaturation in a solution lowered. An expression for the aggregation constant in this equation was proposed. The proposed mechanism of crystal intergrowth duringt bulk crystallization allowed evaluating the specific surface energy of tested salts, which turned out to be in reasonable agreement with published literature data. It was concluded that the intergrowth of crystals during bulk crystallization from solutions proceeded via formation of nucleus‐bridges between crystals. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Practical experimental design techniques for automatic and manual protein crystallization

When crystallizers are searching for the optimum crystallization conditions, they often carry out experiments that are confusing and difficult to interpret. This confusion arises because there are several important variables in any protein crystallization experiment (including protein concentration, precipitant concentration, pH and temperature) and these variables often interact–that is to say, changes in the level of one variable often change the optimum settings of the others. Confusion can be avoided by using appropriate experimental designs where all of the important variables are varied in each experimental run. Some well known and practical designs for automatic and manual crystallization are presented, and a simple practical example is given.     

PCAM: a multi-user facility-based protein crystallization apparatus for microgravity

A facility-based protein crystallization apparatus for microgravity (PCAM) has been constructed and flown on a series of Space Shuttle Missions. The hardware development was undertaken largely because of the many important examples of quality improvements gained from crystal growth in the diffusion-limited environment in space. The concept was based on the adaptation for microgravity of a commonly available crystallization tray to increase sample density, to facilitate co-investigator participation and to improve flight logistics and handling. A co-investigator group representing scientists from industry, academia, and government laboratories has been established. Microgravity applications of the hardware have produced improvements in a number of structure-based crystallographic studies and include examples of enabling research. Additionally, the facility has been used to support fundamental research in protein crystal growth which has delineated factors contributing to the effect of microgravity on the growth and quality of protein crystals.     

Mechanism of crystallization of fast fired mullite-based glass–ceramic glazes for floor-tiles

The mechanism of crystallization from a B₂O₃-containing glass, with composition based in the CaO–MgO–Al₂O₃–SiO₂ system, to a glass–ceramic glaze was studied by different techniques. Glass powder pellets were fast heated, simulating current industrial tile processing methods, at several temperatures from 700 to 1200 °C with a 5 min hold. Microstructural study by field emission scanning electron microscopy revealed that a phase separation phenomenon occurred in the glass, which promoted the onset of mullite crystallization at 900 °C. The amount of mullite in the glass heated between 1100 and 1200 °C was around 20 wt%, as determined by Rietveld refinement. The microstructure of the glass–ceramic glaze heated at 1160 °C consisted of interlocked, well-shaped, acicular mullite crystals longer than 4 μm, immersed in a residual glassy phase.     

In situ data collection and structure refinement from microcapillary protein crystallization

In situ X-ray data collection has the potential to eliminate the challenging task of mounting and cryocooling often fragile protein crystals, reducing a major bottleneck in the structure determination process. An apparatus used to grow protein crystals in capillaries and to compare the background X-ray scattering of the components, including thin-walled glass capillaries against Teflon, and various fluorocarbon oils against each other, is described. Using thaumatin as a test case at 1.8 ? resolution, this study demonstrates that high-resolution electron density maps and refined models can be obtained from in situ diffraction of crystals grown in microcapillaries.     

Enhancement of nucleation during hanging drop protein crystallization using HF Treatment of cover glasses

We examined a simple approach, i.e., etching cover glasses using hydrofluoric acid (HF), to determine whether cover glass treatment enhances nucleation in hanging drop protein crystallization. Hen egg white lysozyme and proteinase K were used as the model proteins. We found that the treatment increased the success rate of crystallization. The results indicated that the simple treatment, which is easy to adopt without changing much in the hanging drop method, can be utilized as an alternative method to enhance protein crystallization screens (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Natural crystallization

Natural mineral crystals grow under a broad spectrum of conditions; from vapors, from hydrothermal solutions, from magmas (high temperature solutions), or through metasomatic or metamorphic reactions. In understanding kinetic problems involved in natural crystallization, there are two ways of approach; (1) experimentally stimulating textures of rocks, and (2) decoding the paragenetic information contained in natural crystals. The latter approach is especially important, since in situ observation is impossible. Key Key features which aid in deciphering natural growth processes and conditions include external forms, surface microtopographs of crystal faces, internal inhomogeneity (growth bands, growth sectors, inclusions, twin or exsolution textures), lattice defects (plane defects, dislocations) and impurities (precipitations). Mainly based on the observations of surface microtopographs of natural crystals, characteristics of crystallization in magma, in hydrothermal solution, in vapor phase, in hydrothermal metasomatism and in regional metamorphism are analysed and reviewed in this paper. The difference and similarity between natural and synthetic crystals are also discussed.     

The effects of microgravity on protein crystallization: evidence for concentration gradients around growing crystals

Atomic force microscopy (AFM) investigations have revealed that macromolecular crystals, during their growth, incorporate an extensive array of impurities. These vary from individual molecules to large particles, and microcrystals in the micron size range. AFM, along with X-ray topology, has further shown that the density of defects and faults in most macromolecular crystals is very high in comparison with conventional crystals. The high defect density is a consequence of the incorporation of impurities, misoriented nutrient molecules, and aggregates of molecules. High defect and impurity density, contributes to a deterioration of both the mechanical and the diffraction properties of crystals. In microgravity, access by impurities and aggregates to growing crystal surfaces is restricted due to altered fluid transport properties. We designed, and have now constructed an instrument, the observable protein crystal growth apparatus (OPCGA) that employs a fused optics, phase shift, Mach–Zehnder interferometer to analyze the fluid environment around growing crystals. Using this device, which will ultimately be employed on the international space station, we have, in thin cells on earth, succeeded in directly visualizing concentration gradients around growing protein crystals. This provides the first direct evidence that quasi-stable depletion zones formed around growing crystals in space may explain the improved quality of macromolecular crystals grown in microgravity. Further application of the interferometric technique will allow us to quantitatively describe the shapes, extent, and magnitudes of the concentration gradients and to evaluate their degree of stability.     

Mechanism and control of crack generation in glass substrates during crystallization of a-Si Films by flash lamp annealing

We investigate the impact of the materials of glass substrates on crack formation during flash lamp annealing (FLA) of 4.5 μm-thick precursor amorphous silicon (a-Si) films for the formation of polycrystalline Si (poly-Si) films. The use of soda lime glass substrates, with the largest thermal expansion coefficient (α) and the lowest glass transition temperature (T_g) in glass materials attempted in this study, results in the serious formation of cracks on and inside the glass substrates. Cracks are also seen on the surface of quartz glass substrates, which have much smaller α and higher T_g, after FLA. Furthermore, flash-lamp-crystallized (FLC) poly-Si films have linearly-connected low-crystallinity regions only when quartz glass substrates are used. These facts indicate that the expansion of Si films induces cracks in quartz glass substrates, while the expansion of the upper part of glass is the cause of the crack formation in glass substrates with large α. The generation of cracks is most significantly suppressed when we use alkali-free glass substrates, with a moderate α and a relatively high T_g, which will contribute to the realization of high-quality poly-Si films and high-performance solar cells.     

Fundamentals of high-temperature crystallization

The fundamental problems of high-temperature crystallization are considered. It is shown that, unlike low-temperature crystallization, high-temperature crystallization proceeds under nonequilibrium conditions, which complicates consideration of related problems.     

Phase-field modeling of glass crystallization: Change of the transport properties and crystallization kinetic

In this work we present many-particle phase-field simulations of primary crystallization with a diffusion coefficient dependent on the local composition of the untransformed matrix. The results show that the experimental kinetics observed in primary crystallization of many metallic systems cannot be described by the soft-impingement effect but to the change of the transport properties of the matrix as the transformation proceeds.