Layered crystals of apo‐ and holoferritin grown by alternating crystallization

We report on the use of alternating crystallization for deposition of layers of different (though closely related) proteins in a single crystal. Investigations were carried out with the unique protein couple consisting of two forms of ferritin, apoferritin and holoferritin from horse spleen, which, despite being of quite different molecular masses, still possess identical organic shells. Crystals of both proteins were used as substrates for subsequent contiguous growth of the partner protein in perfect alignment. We observed continuous growth of combined (onion‐like) single crystals; artificial structures of biological macromolecules can be designed in this way. The homoepitaxial layered growth shows in an unambiguous way that protein crystallization depends only on the surface protein conformation and amino‐acid composition, but not on the internal molecule structure. The limitations of protein crystal growth for designing layered structures of biological macromolecules were revealed by growing of heterogeneous protein crystals onto pre‐existing protein crystalline substrates. Tetragonal crystals of hen egg‐white lysozyme were grown onto cubic apoferritin crystals used as substrates. It was observed that the lysozyme crystals were not lattice‐matched to the ‘host’ apoferritin crystals; this led to mere aggregates of different crystals. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Deuterated hen egg-white lysozyme crystals: Optimization of the growth conditions and morphology

Deuterated and protonated tetragonal lysozyme crystals are grown using the hanging-drop vapor-diffusion method. The size of the lysozyme crystals grown is determined as a function of the concentration of sodium chloride used as a precipitant. It is found that crystallization leads to the formation of lysozyme crystals with three different habits. Morphological and X-ray diffraction analyses of the deuterated and protonated lysozyme crystals demonstrate that, despite the different habits, all the crystals grown belong to the tetragonal crystal system. The simple forms of lysozyme crystals are revealed. It is shown that the habits of the lysozyme crystals are determined by the specific combinations of simple forms. The mechanisms responsible for the formation of lysozyme crystals with different habits are discussed.     

Protein crystal growth on the Russian segment of the International Space Station

Experiments on protein crystallization on the Russian segment of the International Space Station were started in 2005. These experiments were performed in the Modul’-1 protein crystallization apparatus specially designed for crystal growth by the free-interface-diffusion method. This paper describes experiments on the crystallization of lysozyme, carboxypeptidase B, and recombinant human insulin on Earth and in microgravity using the Modul’-1 protein crystallization apparatus during the ISS-11-ISS-14 space flights. Crystals of all proteins grown in microgravity have larger sizes than those grown on Earth. Space-grown crystals of lysozyme and insulin characterized by X-ray diffraction were shown to diffract to higher resolution than the Earth-grown crystals. The three-dimensional structures of Zn-insulin crystals grown both on Earth and in microgravity were established. The conformation of the Zn-insulin hexamer in the crystalline state is described.     

X-ray topography of a crystal of tetragonal lysozyme

A crystal of the tetragonal modification of lysozyme grown under controlled conditions with in situ monitoring of the growth kinetics and morphology of the (110) face is studied by X-ray topography using synchrotron radiation. The choice of a diffraction reflection with optimal dispersion allows us to obtain an informative X-ray topographic image of the sample. It is found that the striations that are formed under changing supersaturation correspond to the type of zonality characteristic of crystals growing by the normal mechanism and differ from those observed in inorganic crystals growing by the layer-by-layer mechanism.     

熔盐法和水热法KTP晶体的晶胞参数及拉曼光谱特征

利用X射线衍射和显微激光拉曼光谱研究熔盐法自发结晶的KTP晶体、顶部籽晶熔盐法KTP晶体和水热法KTP晶体的晶胞参数和拉曼光谱特征,分析和比较不同方法生长的KTP晶体的晶体结构与化学键特征峰.研究表明:KTP晶体的晶胞参数与晶体生长方法有关,熔盐法自发结晶的KTP晶体生长过程中降温速率较快,晶胞体积相对较小;熔盐法和水热法KTP晶体中部分拉曼特征峰的位置因生长方法不同呈现一定的差异,水热法KTP晶体在782 cm-1、744 cm-1和515 cm-1处出现的特征峰可视为水热法KTP晶体的标志峰,借此可将其与熔盐法晶体相区分.     

A more clear insight of the lysozyme crystal composition

Crystallization can be used as a purification method for proteins. Lysozyme was chosen as a model substance. Changing crystallization conditions will lead as shown to different lysozyme crystal morphologies with different properties. Beside others, lysozyme crystals can show a Tetragonal, High Temperature and Low Temperature Orthorhombic crystal morphology. Experiments such as conductivity measurements, pH tests, chloride detection tests, experiments using methylene blue as a dye and dissolution experiments were carried out to investigate the composition of the lysozyme crystals. It is proven that lysozyme crystals are made up of the initial buffer solution components: lysozyme (the protein), water which is part of the crystal lattice, salt ions which are attached to the protein molecule and voids filled with the buffer solution containing the crystallization agent (e.g. salt). Interesting dissolution behaviours of the lysozyme crystals were observed which are not described so far elsewhere (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Crystalline quality of lysozyme crystals grown in agarose and silica gels studied by X-ray diffraction techniques

The crystalline quality of hen egg white lysozyme (HEWL) crystals grown in agarose gels and in silica gels has been characterized by measuring resolution and mosaic spread. These crystals have been compared to solution grown ones. A quasi-plane-wave X-ray topography study has also been done on some crystals. The study concerns triclinic, monoclinic, orthorhombic and tetragonal forms of HEWL. One observes that the resolution is not really changed by gelling the growth medium, even for rather high gel contents (agarose 0.5% wt/wt). On the contrary, mosaicity, characterized through reflection profile recordings allows to differentiate crystals grown by different techniques: agarose gel grown crystals are, on average, better than solution grown ones but the best crystals are obtained in silica gel. X-ray topography confirms this result.     

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.     

Direct AFM observations of impurity effects on a lysozyme crystal

Impurity effects on the growth of tetragonal lysozyme crystals have been studied using in situ atomic force microscopy. Commercially available hen egg white lysozyme was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis with silver staining, and purified by re-crystallization and successive high pressure liquid chromatography. On the (1 1 0) crystal surface, there was no significant difference in morphology between crystals grown in commercial and in purified solutions. On the (1 0 1) surface, however, a large number of small particles were found when the crystal was grown in the commercial solution, while the surface grown in the purified solution was quite smooth. Among the typical residual impurities contained in commercial lysozyme, only covalently bound lysozyme dimer yielded such particles. From measurements of particle separation and an estimate of the critical nucleation size, we infer that the particles reduced the step velocity according to the mechanism described by Cabrera et al. [N. Cabrera, D.A. Vermilyea, in: R.H. Doremus et al. (Eds.), Growth and Perfection of Crystals, 1958, P. 393].     

Specific features in shaping Bi<Subscript>12</Subscript>GeO<Subscript>20</Subscript> crystals grown by low thermal gradient Czochralski technique

Germanosillenite (BGO) crystals have been grown by the low thermal gradient Czochralski technique [1] at crystallization rates of v = 0.05–4 mm/h. The evolution regularities of the faceted front forms have been studied taking into consideration their growth conditions (crystallization rate and thermal conditions). The orientations of the faces forming the crystallization front during crystal growth in the 〈111〉 direction have been determined. The relationship between the front morphology (and, therefore, growth conditions) and the quality of crystals formed is established. The quality of the BGO crystals grown is evaluated by X-ray topography.     

Spatiotemporal protein crystal growth studies using microfluidic silicon devices

Fundamental investigations of protein crystallization using miniaturized microfluidic silicon devices were presented towards achieving spatiotemporal nucleation and subsequent post-nucleation growth. The developed microfluidic silicon device was typically composed of crystal growth cell, reservoir cell, and optionally of heater elements for supersaturation control. A specific fine pattern area in the growth cell which was fabricated on the silicon substrate with doped p- and n-type silicon layers, served as spatially selective nucleation site of dissolved protein molecules through electrostatic attractive force. In a model material, hen egg white lysozyme, a large number of crystals were grown on the defined nucleation site evenly spaced from each other, whereas parasitic crystal growth positioned around the selective nucleation site, was suppressed by the effects of electrostatic repulsive force between the doped silicon surface and charged protein molecules. A possible crystallization mechanism of describing the heterogeneous nucleation during the initial stage and during the growth of the crystal at the electrolyte–semiconductor silicon surface is proposed and discussed.     

Perfection and homogeneity of space-grown GaSb:Te crystals

A Te-doped GaSb crystal grown by the method of directed crystallization under microgravitation has been studied by X-ray topography. It is shown that the structural perfection of the crystal part grown without contact with the ampule walls is substantially higher than the structural perfection of the crystals grown under terrestrial conditions. The distribution of a Te dopant in the crystal is studied by quantitative X-ray topography, and it is shown that, if the gap between the growing crystal and the ampule is small, the Marangoni convection in the melt can be less intense than the convection provided by residual microgravitation. The relation between the formation of a flat face on the crystallization front (faceting) and the formation of twins is also established.     

Structural characterization of heteroepitaxial films of CdSe and ZnTe

The effects of composition, orientation and temperature of substrate on the structure and morphology of epitaxial CdSe and ZnTe films are analyzed. Single crystal wafers of zinc telluride and zinc selenide oriented along (110) and natural cleaved mica were used as substrates. This paper presents a complex investigation of the growth mechanism and crystal perfection of the films by optical microscopy, X-ray and electron diffraction, and by electron microscopy. The structure of the transient region of the film substrate interface was studied by X-ray microanalyzer and by scanning electron microscope. Cadmium selenide layers grown on mica had hexagonal structure and were oriented in the (0001) plane, those grown on zinc telluride crystals had cubic structure and orientation along (110). Zinc telluride layers were cubic and were oriented parallel the (111) plane when deposited on mica, and parallel to (110) when deposited on zinc selenide substrates. It is shown that the layers grew by a layer mechanism. Epitaxial growth of cubic cadmium selenide layers on zinc telluride single crystals, as well as of ZnTe layers on ZnSe substrates, made it possible to grow heterojunctions with a low density of misfit dislocations at the interface; this gave rise to intensive injection electroluminescence and effective separation of non-equilibrium charge carriers in such structures.     

Small-angle X-ray scattering study of conditions for the formation of growth units of protein crystals in lysozyme solutions

The structural composition of lysozyme solutions favorable for the formation of the tetragonal form of protein crystals was studied by synchrotron-based small-angle X-ray scattering depending on the protein concentration and the temperature. Along with lysozyme monomers, dimers and octamers are found in crystallization solutions; the octamer content increases with an increase in the protein concentration.     

The crystallization of lysozyme in the system of ionic liquid [BMIm][BF4]‐water

Lysozyme crystallization was conducted in the ionic liquid (IL) 1‐butyl‐3‐methylimidizolium tetrafluoroborate ([BMIm][BF₄]) with different buffer/IL proportions. It was found that the addition of [BMIm][BF₄] could promote the crystallization process, during which more lager single crystals with controllable morphologies could be obtained due to the manageable crystal growth velocity. A probable explanation was proposed based on the influence of the ionic polarization and kinetics in the lysozyme crystallization. Moreover, the transform in coordination number and the relative growth rate of different crystal faces were discussed. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

PVT法同质外延制备大尺寸Al极性氮化铝晶体生长工艺及其表征分析研究

本文基于自主设计的氮化铝生长炉,开展了四组不同工艺条件下Al极性面氮化铝籽晶同质外延生长氮化铝单晶的生长特征及其结晶质量表征研究。研究发现:不同工艺条件下生长的晶体的拉曼图谱E₂(high)特征峰峰位表明,晶体内部均存在较小的拉应力;在坩埚顶部在相对较高温度2 210 ℃、坩埚底部与顶部温差42 ℃的低过饱和度生长条件下,晶体表面光滑,呈现阶梯流生长形貌,并具有典型的氮化铝单晶生长习性面,晶体初始扩张角大于40°,高分辨率X射线衍射(HRXRD)测得0002、1012反射摇摆曲线及拉曼光谱检测结果表明,该条件下生长的氮化铝晶体结晶质量优异,并可实现快速扩径。基于该生长条件,通过外延生长后成功获得尺寸ϕ45~47 mm的氮化铝单晶锭,相关表征结果表明生长的氮化铝晶体具有优越的结晶性能。     

Growth of KTiOPO<Subscript>4</Subscript> crystals doped with zinc and studies of their physical properties and specific structural features

A series of potassium titanyl phosphate single crystals doped with zinc (KTP: Zn) is grown by spontaneous flux crystallization. Their properties and the way the additive is implanted in the crystal lattice are studied. The inclusion of zinc atoms in the KTP structure is evidenced by the data of chemical analysis and the results of studies of electrophysical properties (the growth of conductivity and increase of relaxation anomalies). Precision X-ray diffraction studies of KTP: Zn single crystals reveal changes in the channel of the structure which correlate with the physical properties of this crystal series. No substitution of zinc atoms for titanium, phosphorus, or potassium atoms is found in the structure. Zinc atoms can be located at structural defects, domain walls, and the crystal surface.     

In situ study of the state of lysozyme molecules at the very early stage of the crystallization process by small-angle X-ray scattering

The molecular state of hen egg white lysozyme in solution has been studied by small-angle X-ray scattering (SAXS) combined with molecular simulation. The addition of a precipitant is shown to change the state of the protein molecules in solution. The SAXS data were processed using the constructed models of different oligomers. Under the crystallization conditions, lysozyme is shown to be present in solution as monomers (96.0%), dimers (1.9%), and octamers (2.1%), whereas tetramers and hexamers are not found. The modeled structure of the octamer is not consistent with the commonly accepted unit cell containing eight lysozyme molecules. Meanwhile, the modeled octamers are well-fitted to the crystal structure and can serve as building blocks in the course of crystal growth.     

Implementation of Temperature-Controlled Method of Protein Crystallization in Microgravity

An experimental scientific equipment for implementing temperature-controlled protein crystallization in capillaries under microgravity has been developed, fabricated, and tested. This crystallization method, providing on-line separate control of crystal growth both in the stage of nucleation of crystals and during their further growth, requires small amounts of protein solution. The equipment has been tested on board of Foton-M4 spacecraft (growth of lysozyme protein crystals of high structural quality in microgravity) using a cyclogram developed in ground-based experiments. The results obtained have demonstrated efficiency and importance of the developed equipment and method for growing biomacromolecular crystals of high-structural quality.

     

Gel growth of α and γ glycine and their characterization

The microbial free single crystals of α and γ glycine were grown from gel at room temperature in a new chemical route. These crystals showed a superior quality than the solution grown crystals. The metastable α-form and the stable γ-form of glycine were crystallized in silica gel by solubility reduction method. The form of crystallization is confirmed by single crystal and powder X-ray diffraction analyses. The crystals of α and γ glycine were found to crystallize in monoclinic and hexagonal crystal systems, respectively. For analyzing the functional group and thermal stability of α and γ glycine crystals, spectroscopic and thermal analyses have been carried out. The dielectric studies were performed to find the dielectric constant of the grown crystals and the results are discussed. Second harmonic generation efficiency of the crystal was measured by Kurtz’s powder method using Nd:YAG laser and it was found to be 2.68 times that of potassium dihydrogen phosphate crystals.