A short overview on practical techniques for protein crystallization and a new approach using low intensity electromagnetic fields

This contribution deals with a practical overview of some popular and sophisticated crystallization methods that help increase the success rate of a crystallization project and introduces a newly developed method involving low intensity electromagnetic fields. Aiming to suggest a methodology to follow, the present contribution is divided into two main parts in a logical order to get the best crystals for high resolution X-ray crystallographic analysis. The first part starts with a short review of the chemical and physical fundamentals of each crystallization method through different strategies based on physicochemical approaches. Then, practical non-conventional techniques for protein crystallization are presented, not only for growing protein crystals, but also for controlling the size and number of crystals. These include crystal growth in gels, counter-diffusion, seeding, and macromolecular imprinted polymers (MIPs). The second part shows the effects of coupling low intensity electric fields (in the scale of units of  μAmperes) with weak magnetic fields (in the scale of milli Tesla) applied to protein crystallization. This approach consists of a novel experimental set up, which was used to study the influence of the coupled fields on the crystallization of lysozyme in solution and in gel media. This new approach is based on the classical theories of transport phenomena and offers a more accessible strategy to obtain suitable crystals for X-ray characterization or Neutron diffraction investigations.     

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)     

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.     

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.     

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.     

Mathematical modeling and experimental investigation of the effect of temperature gradients on crystallization processes under terrestrial and space conditions

Mathematical modeling of the processes of heat and mass transfer during directed crystallization under terrestrial and space conditions is performed on the basis of experimental data on the temperature distribution (boundary conditions). Convective processes are described by the system of Oberbeck-Boussinesq equations together with the heat-conduction equation (the Stefan problem). A dependence of the intensity of thermal gravitational convection on the radial and axial temperature gradients is established. It is shown that one of the necessary conditions for the growth of homogeneous semiconductor crystals under both terrestrial and zero-gravity (on board spacecraft) conditions is the absence of the free surface of a melt (the Marangoni convection) and optimization of the temperature gradients (first of all, the radial gradient).     

Modeling of the shape of polyethylene oxide single crystals and determination of the kinetic crystallization parameters: Theoretical and experimental approaches

The curvature of faces of polymer single crystals is described by the system of Mansfield equations, which is based on the Frank-Seto growth model. This model assumes the velocity of nucleus steps to be the same for their propagation to the right and left and is valid only for symmetric crystallographic planes. To describe the shape of polyethylene oxide single crystals grown from melt and limited by the {100} and {120} folding planes, it is assumed that the layer velocities to the right and left are different on {120} faces. This approach allows modeling, with a high accuracy, of the observed shapes of polymer single crystals grown at different temperatures, which makes it possible to determine unambiguously the fundamental crystallization parameters: the dimensionless ratio of the secondary homogeneous nucleation rate to the average velocities of nuclei along the crystallization planes and the ratio of nucleus velocities to the right and left. In addition, it was found that a known macroscopic single-crystal growth rate can be used to determine the absolute values of the secondary homogeneous nucleation rate and the velocities of nuclei along the growth plane.     

Pressure and concentration dependence of nucleation kinetics for crystallization of subtilisin

Nucleation kinetics of subtilisin was studied as a function of supersaturation (10–70 mg/ml) and pressure (0.1–34 MPa). The nucleation was found to have a 1.6 order dependence on supersaturation. Extent of nucleation decreased by a factor of 60 as the pressure increased from 0.1 to 34 MPa. High pressure was also used as a probe to understand the nucleation behavior of subtilisin and an estimation of the pressure-dependence of nucleation rate provided a value of 330 cm³/mol for the activation volume for nucleation. The residues in the contact region were determined to be hydrophilic by protein structure analysis. Responsible mechanisms are hypothesized.     

Comparison between approaches for the experimental determination of metastable zone width: A case study of the batch cooling crystallization of ammonium oxalate in water

In situ ATR-FTIR spectroscopy coupled with in situ image acquisition measurements were used to determine the solubility curve and to investigate the metastable zone width (MSZW) of ammonium oxalate monohydrate (AO) aqueous solutions under polythermal conditions. The experimental data allowed estimating the MSZW and induction time of the system and the results were compared with published literature values for the same system [1]. “Pseudo” induction time estimation techniques based on polythermal methods showed that AO aqueous solutions exhibit two nucleation regimes depending on the cooling rates.Even though they are based on rough and questionable assumptions, induction time and MSZW estimation methods are often considered as essential for the development of industrial crystallization processes. However, both the relevancy and the physical meaning of the results provided by these methods are uncertain. In the actual industrial context where many advanced measurement techniques and modeling tools became available, the present paper intends to call into question the outcome of the notions of MSZW and induction time.     

Diffraction techniques and structural models for amorphous materials

The need to use theoretical models to represent the structure of amorphous substances poses questions about the reliability of the experimental reference data against which these models are tested. An understanding of the real situation in the case of X-ray diffraction curves has been attempted. Through an empirical approach, based on the examination of five independent X-ray diffraction measurements taken on the same sample of amorphous silica, it is concluded that this technique is a selective filter for the various structural hypotheses.     

Advancements (and challenges) in the study of protein crystal nucleation and growth; thermodynamic and kinetic explanations and comparison with small-molecule crystallization

This paper reviews advancements and some novel ideas (not yet covered by reviews and monographs) concerning thermodynamics and kinetics of protein crystal nucleation and growth, as well as some outcomes resulting therefrom. By accounting the role of physical and biochemical factors, the paper aims to present a comprehensive (rather than complete) review of recent studies and efforts to elucidate the protein crystallization process. Thermodynamic rules that govern both protein and small-molecule crystallization are considered firstly. The thermodynamically substantiated EBDE method (meaning equilibration between the cohesive energy which maintains the integrity of a crystalline cluster and the destructive energies tending to tear-up it) determines the supersaturation dependent size of stable nuclei (i.e., nuclei that are doomed to grow). The size of the stable nucleus is worth-considering because it is exactly related to the size of the critical crystal nucleus, and permits calculation of the latter. Besides, merely stable nuclei grow to visible crystals, and are detected experimentally. EBDE is applied for considering protein crystal nucleation in pores and hydrophobicity assisted protein crystallization. The logistic functional kinetics of nucleation (expressed as nuclei number density vs. nucleation time) explains quantitatively important aspects of the crystallization process, such as supersaturation dependence of crystal nuclei number density at fixed nucleation time and crystal size distribution (CSD) resulting from batch crystallization. It is shown that the CSD is instigated by the crystal nucleation stage, which produces an ogee-curve shaped CSD vs. crystal birth moments. Experimental results confirm both the logistic functional nucleation kinetics and the calculated CSD. And even though Ostwald ripening modifies the latter (because the smallest crystals dissolve rendering material for the growth of larger crystals), CSD during this terminal crystallization stage retains some traces of the CSD shape inherited from the nucleation stage. Another objective of this paper is to point-out some biochemical aspects of the protein crystallization, such as bond selection mechanism (BSM) of protein crystal nucleation and growth and the effect of electric fields exerted on the process. Finally, an in-silico study on crystal polymorph selection is reviewed.     

Application of D‐optimal experimental design in nano‐sized ZSM‐5 synthesis for obtaining higher crystallinity

D‐optimal experimental design with three levels of SiO₂/Al₂O₃, template/SiO₂, H₂O/SiO₂, SiO₂/Na₂O and TPABr/TPAOH ratio parameters was used to optimize the experimental parameters by the analysis of variance (ANOVA). The effects of above mentioned ratios in the initial synthetic mixture on the crystallinity of the ZSM‐5 zeolite were studied. The synthesized samples were characterized by XRD, FE‐SEM, and TEM analysis. Fischer test results showed that SiO₂/Al₂O₃ and H₂O/SiO₂ molar ratios are the most and least effective parameters, respectively, in the range studied. The most important two‐way interaction variable was that of template/SiO₂ and Na₂O/SiO₂ molar ratios. The optimum composition of the gel compound to achieve relative maximum crystallinity is SiO₂/Al₂O₃ = 99.96, template/SiO₂ = 0.16, H₂O/SiO₂ = 34.68, Na₂O/SiO₂ = 0.02 and TPABr/TPAOH = 1.44.     

Materials and fabrication techniques for integrated optics: Organic and amorphoys materials

In parallel with the development of device concepts in integrated optics, there has been a comparable advance in material systems and fabrication technology enabling rather more sophisticated devices to be realised. This review covers examples of work at University College London on amorphous and organic material deposition techniques specifically developed to produced high optical quality is presented as an illustration of the advances in shaping technology now being used in integrated optics.     

Full-wafer mapping and response surface modeling techniques for thin film deposition processes

Computational techniques for representing and analyzing full-wafer metrology data are developed for chemical vapor deposition and other thin-film processing applications. Spatially resolved measurement data are used to produce “virtual wafers” that are subsequently used to create response surface models for predicting the full-wafer thickness, composition, or any other property profile as a function of processing parameters. Statistical analysis tools are developed to assess model prediction accuracy and to compare the relative accuracies of different models created from the same wafer data set. Examples illustrating the use of these techniques for film property uniformity optimization and for creating intentional film-property spatial gradients for combinatorial CVD applications are presented.     

Role of analytical techniques for characterisation of advanced and high purity materials

High purity metals and other advanced materials are extensively employed in various applications connected with electronics, solar energy conversion, superconductors, shape memory alloys and other industrial applications of importance. In many of these applications, the properties are affected by the nature and concentration of impurities present in the material. The use of modern analytical techniques for chemical and surface characterization is highly beneficial for the evaluation of such materials for various critical applications. The sophisticated chemical analysis techniques like AAS, GFAAS, ICPAES, ICPMS, NAA and DPASV helps in chemical characterization which together with surface analytical techniques like XPS provides complete evaluation of the material for various applications.     

Fractal slice model analysis for effective thermal conductivity and temperature distribution of porous crystal layer via layer crystallization

A fractal slice model was established based on the reported model to predict the effective thermal conductivity of a porous crystal layer via layer crystallization. The temperature distribution of the crystal layer was obtained by the fractal slice method. The simulation results agreed with the experimental data better than the other theoretical models. The results were helpful to enhance the thermal transport by mitigating the thermal resistance effectively.     

Critical considerations on the Augis and Bennett method for evaluating the crystallization activation energy by means of non-isothermal data

The problems resulting from deriving the equation underlying the Augis and Bennett method (J. A. Augis, J. E. Bennett, J. Therm. Anal., 13 (1978) 183) often used in assessing the crystallization process activation energy (E), are critically analyzed. Solving such problems has resulted in a new iterative procedure for the E evaluation by making use of the non-isothermal data recorded at some heating rates. The theoretical results were checked for some experimental data given in late literature.     

A combination of oscillating slit and oscillating film techniques for the investigation of lattice defects

A combination of oscillating slit and oscillating film techniques is described. This X-ray topographical method allows reflections due to the same places on the crystal surface to be recorded first in form of a topogramme registrated on the oscillating film set in the parallel position to the crystal surface. Second on the stationary film set parallel to the crystal surface in the form of an interference line Kα₁ or Kß outgoing from the investigated area and registrated on the first topogramme at the fixed angular position of the crystal and slit and next, recorded separate on the oscillating film and on the stationary film set in the distance of 50 mm from the crystal (oscillation axis). As an example a serie of photographs corresponding to four types of pattern obtained in the case of a PbSnTe crystal with small angle boundaries are presented.