Crystal growth of phosphopantetheine adenylyltransferase,carboxypeptidase t,and thymidine phosphorylase on the international space station by the capillary counter-diffusion method

Crystals of phosphopantetheine adenylyltransferase from Mycobacterium tuberculosis, thymidine phosphorylase from Escherichia coli, carboxypeptidase T from Thermoactinomyces vulgaris and its mutant forms, and crystals of complexes of these proteins with functional ligands and inhibitors were grown by the capillary counter-diffusion method in the Japanese Experimental Module Kibo on the International Space Station. The high-resolution X-ray diffraction data sets suitable for the determination of high-resolution three-dimensional structures of these proteins were collected from the grown crystals on the SPring-8 synchrotron radiation facility. The conditions of crystal growth for the proteins and the data-collection statistics are reported. The crystals grown in microgravity diffracted to a higher resolution than crystals of the same proteins grown on Earth.     

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.     

Crystallogenesis of biological macromolecules. Biological, microgravity and other physico-chemical aspects

After an historical introduction and justification of the importance of proteins (as well as other macromolecules or macromolecular assemblies of biological origin) in modern biology but also in physics, this review presents the state of the field of macromolecular crystallogenesis. The basic questions underlying the crystallization of macromolecules will be addressed and discussed in a first part. The still unsolved problems are highlighted. This section also discusses the methodological approaches that can be used. In the second part, some of the recent achievements in the field are presented. We show how physical methods have contributed to a deeper understanding of protein crystallization. Emphasis is given to the parameter microgravity, since the projects concerning crystallizations in this environment have stimulated the physico-chemical research in the field. Finally, the future perspectives are outlined.     

Results of crystal growth of bismuth-antimony alloys (Bi100–xSbx) in a microgravity environment

Results of two experiments are presented for growth of crystals from (Bi_100–xSb_x) alloys in a microgravity environment. In the growth experiments different variants of the Bridgman technique were used. It was shown that in crystal growth from the melt in closed ampoules under microgravity conditions convection can be prevented completely. Therefore it is possible to grow crystals from melts of some components under diffusion controlled conditions of mass transfer. In microgravity a reduced interaction between the melt and the confining walls was observed even if they have large contact with each other. The investigation of surface morphology corroborated the importance of surface effects for crystal growth from the melt under microgravity conditions. Measurements of electronic properties of crystals grown in microgravity showed a good quality in comparison to earth grown crystals. Because under microgravity conditions in closed ampoules the diffusion controlled mass transfer can be realized and the interaction between the melt and confining wall is reduced, homogeneous crystals with high perfection can be grown melts of some components.     

High-Quality Protein Crystal Growth of Mouse Lipocalin-Type Prostaglandin D Synthase in Microgravity

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) catalyzes the isomerization of PGH(2) to PGD(2) and is involved in the regulation of pain and of nonrapid eye movement sleep and the differentiation of male genital organs and adipocytes, etc. L-PGDS is secreted into various body fluids and binds various lipophilic compounds with high affinities, acting also as an extracellular transporter. Mouse L-PGDS with a C65A mutation was previously crystallized with citrate or malonate as a precipitant, and the X-ray crystallographic structure was determined at 2.0 ? resolution. To obtain high-quality crystals, we tried, unsuccessfully, to crystallize the C65A mutant in microgravity under the same conditions used in the previous study. After further purifying the protein and changing the precipitant to polyethylene glycol (PEG) 8000, high-quality crystals were grown in microgravity. The precipitant solution was 40% (w/v) PEG 8000, 100 mM sodium chloride, and 100 mM HEPES-NaOH (pH 7.0). Crystals grew on board the International Space Station for 11 weeks in 2007, yielding single crystals of the wild-type L-PGDS and the C65A mutant, both of which diffracted at around 1.0 ? resolution. The crystal quality was markedly improved through the use of a high-viscosity precipitant solution in microgravity, in combination with the use of a highly purified protein.     

分离结晶垂直Bridgman法生长CdZnTe晶体的全局数值模拟

采用FLUENT软件对分离结晶Bridgman法生长CdZnTe晶体进行了全局数值模拟.模拟对象为:熔体上部边界条件分别为固壁和自由表面时两种晶体生长系统.重点考虑坩埚和晶体之间狭缝宽度e和重力对分离结晶过程的影响.在计算中分别取e=0 mm、0.5 mm和1 mm三种狭缝宽度,得到了在微重力和常重力条件下的温度分布、结晶界面形状以及流函数分布图.结果表明:在微重力条件下,当熔体上部为固壁时,随着狭缝宽度的增大,热毛细力作用增强,流动强度增强;当熔体上部为自由表面时,则与之相反.在常重力条件下,由于浮力-热毛细对流的共同作用,随着狭缝宽度的增加,流动强度逐渐减弱,有助于提高晶体生长质量.     

Lower dimer impurity incorporation may result in higher perfection of HEWL crystals grown in microgravity: A case study

Crystals of tetragonal hen egg white lysozyme (HEWL) grown on a series of space missions and their terrestrial counterparts were analyzed by gel electrophoresis and X-ray diffraction. The crystals were produced by vapor-diffusion and dialysis methods. The microgravity and terrestrial grown HEWL crystals were found to have effective partitioning coefficients (K_eff) for an oxidatively formed covalent dimer impurity (MW 28 K) of 2 and 9, respectively, i.e. the latter contain 4.5 times more dimers. The microgravity grown crystals allowed the collection of 24% more useful reflections and improved the resolution from 1.6 to 1.35 Å. Other improvements were also noted including lower isotropic B-factors of 16.9, versus 23.8 Å² for their terrestrial counterparts. High-resolution laser interferometry was applied quantitatively to evaluate the influence of dimer impurity on growth kinetics. It is shown that the growth of the (1 0 1) face from solution into which 1% dimers were introduced decelerates with increasing solution flow rate and the growth stops at a flow rate of about 0.2 mm/s. This effect occurs faster than in ultrapure solutions. The covalently bound dimers essentially increase the amplitudes of the striation-inducing growth rate fluctuations. The effect is ascribed to the enhanced transport of growth inhibiting HEWL dimer to the interface. Theoretical analysis shows that a stagnant solution around a growing crystal is strongly depleted with respect to impurity by about 60% for the measured growth parameters as compared to the solution bulk. Thus, a crystal in microgravity grows from essentially purer solution than the ones in the presence of convection flows. Therefore, it traps less stress inducing impurity and should be more perfect. For crystal/impurity systems where K_eff is small enough microgravity should have an opposite effect.     

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.     

Gravitational Effects on Mixing and Growth Morphology of an In0.5Ga0.5 System

A mixing experiment of multicomponents melts was performed using a uniform temperature furnace in the Second International Microgravity Laboratories (IML-2) mission. Growth morphologies and Ga concentration profiles were analyzed for the samples with the compositional ratio of 0.5 In–0.5 Ga–1.0 Sb grown under microgravity and on earth. The sample with free surface grown under microgravity was nearly spherical in shape, except some parts with projections. Ga was dispersed homogeneously in the bulk because the mixing was enhanced by Marangoni convection due to the concentration gradient. On the other hand, the sample grown on earth was a double cylindrical shape with different diameters, and Ga concentration decreased from top to bottom, showing clearly the effect of gravity. Many needle crystals were formed in both space and earth samples due to rapid cooling. The average size of the needle crystals grown in space was larger than that of the earth sample.     

Three-dimensional structure of phosphoribosyl pyrophosphate synthetase from <Emphasis Type="Italic">E. coli</Emphasis> at 2.71 Å resolution

Phosphoribosyl pyrophosphate synthetase from Escherichia coli was cloned, purified, and crystallized. Single crystals of the enzyme were grown under microgravity. The X-ray diffraction data set was collected at the Spring-8 synchrotron facility and used to determine the three-dimensional structure of the enzyme by the molecular-replacement method at 2.71 Å resolution. The active and regulatory sites in the molecule of E. coli phosphoribosyl pyrophosphate synthetase were revealed by comparison with the homologous protein from Bacillus subtilis, the structure of which was determined in a complex with functional ligands. The conformations of polypeptide-chain fragments surrounding and composing the active and regulatory sites were shown to be identical in both proteins.     

Minimum volume stability limits for axisymmetric liquid bridges subject to steady axial acceleration

In this paper the influence of an axial microgravity on the minimum volume stability limit of axisymmetric liquid bridges between unequal disks is analyzed both theoretically and experimentally. The results here presented extend the knowledge of the static behaviour of liquid bridges to fluid configurations different from those studied up to now (almost equal disks). Experimental results, obtained by simulating microgravity conditions by the neutral buoyancy technique, are also presented and are shown to be in complete agreement with theoretical ones.     

Growth of Zn‐doped Germanium under Microgravity

The doping of germanium with zinc from a remote, temperature‐stabilized source was studied under microgravity. A nominally undoped Ge‐crystal was grown by the Gradient‐Freeze technique with the melt surface being in permanent contact with a gaseous atmosphere of zinc. The dopant and carrier concentrations in the solidified Germanium were measured by SIMS, Hall and resistance measurements and compared with the results of a terrestrial reference experiment as well as with concentration profiles calculated on the basis of the thermodynamics of the growth system. The results prove the possibility of vapour phase doping under microgravity. Moreover, the Zn‐concentration at the initial phase boundary even agrees well with the equilibrium value, strongly indicating a nearly homogeneous distribution of the dopant within the melt before the crystallization.     

Synthesis and characterization of large single crystals of NpPd3 by flux method

Synthesis of the active [Ni–Fe]-hydrogenase in prokaryotes requires a series of ancillary maturation factors. Among them, the HypF maturation factor is a multidomain 82 kDa protein, whose N-terminal domain displays sequence and structural similarities to acylphosphatases. Acylphosphatases are small enzymes that are able to catalyze carboxyl-phosphate bond hydrolysis in acylphosphates, as well as in nucleoside di- and tri-phosphates and in arylphosphates. Here, we present a crystallographic comparison between microgravity and earth-grown crystals of the HypF N-terminal domain. Both crystals were of excellent quality, thereby allowing us to collect very high resolution datasets. A detailed analysis of data collection and refinement statistics, together with an analysis of the diffraction pattern showed that microgravity would appear to further improve the internal order of crystals.     

移动加热器法生长碲锌镉晶体的组分输运与界面形貌研究

移动加热器法(THM)生长碲锌镉晶体时,界面稳定性对晶体生长的质量有很大影响。本文基于多物理场有限元仿真软件Comsol建立了THM生长碲锌镉晶体的数值模拟模型,讨论了Te边界层与组分过冷区之间的关系,对不同生长阶段的物理场、Te边界层与组分过冷区进行仿真研究,最后讨论了微重力对物理场分布的影响,并对比了微重力与正常重力下的生长界面形貌。模拟结果表明,Te边界层与组分过冷区的分布趋势是一致的,在不同生长阶段,流场中次生涡旋的位置会发生移动,从而导致生长界面的形貌随着生长的进行发生变化,同时微重力条件下形成的生长界面形貌最有利于单晶生长。因此,在晶体生长的中前期,对次生涡旋位置的控制和对组分过冷的削弱,是THM生长高质量晶体的有效方案。     

Concentration distribution in crystallization from solution under microgravity

Concentration distribution in crystallization from solution under microgravity is numerically studied. A quasi-steady state growth and dissolution in a 2D rectangular enclosure filled with sodium chlorate (NaClO₃) aqueous solution, in which one wall is the growth surface of the crystal and the opposite one is the dissolution surface, is considered. The solute transport process at the growth surface is described by the diffusion-reaction theory with finite interface kinetics coefficient. The results show that the concentration at the growth surface is supersaturated and the supersaturation distribution is of non-uniformity, i.e. the supersaturation in a region facing an incoming flow is high. On the other hand, the non-uniformity of supersaturation at the growth surface is closely related to the gravity level even under microgravity, it exponentially increases as the thermal Rayleigh number on behalf of the gravity level rises.     

Comment on the paper “Contribution to the stability analysis of the dewetted Bridgman growth under microgravity conditions”

New considerations of the stability problem of dewetting, as addressed in the initial paper [Cryst. Res. Technol. 39 , 491 (2004)], show that, in the most general case, when dewetting occurs in microgravity it is necessarily stable. This explains why the phenomenon has been observed so many times during solidification of semiconductors in space. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

A crystal of a typical EF-hand protein grown under microgravity diffracts X-rays beyond 0.9 Å resolution

We report on our recent observation that crystals of a typical EF-hand protein (parvalbumin or Pa; Ca-loaded component from pike muscle with isoelectric point 4.10) grown under microgravity conditions diffract X-rays to a resolution better than 0.9 Å. The crystals were grown in the US space shuttle and characterized at 100 K, using an X-ray synchrotron beam. An effective atomic resolution has been achieved and substates in the conformation of the protein are observed. Large crystals up to 3 mm were also obtained.     

Numerical study to investigate the effect of partition block and ambient air temperature on interfacial heat transfer in liquid bridges of high Prandtl number fluid

Surface heat transfer at the liquid–air interface in liquid bridges of high Prandtl number fluid is known to affect the transitional characteristics appreciably. The heat transfer characteristics under microgravity conditions become much different from those of normal gravity mainly due to the absence of natural convection. The present study deals with numerical computations of flow and heat transfer characteristics in the liquid and surrounding air and also at the liquid–air interface of thermocapillary flow in liquid bridges of high Prandtl number fluid. The governing equations are solved in the coupled domain of the liquid bridge and the surrounding air with the help of available commercial CFD software. The results obtained for a range of Marangoni numbers indicate that by placing a partition block in the air region under normal gravity conditions, the surface heat transfer characteristics of microgravity conditions could be effectively mimicked. The effect of ambient temperature on the surface heat transfer has also been investigated and it has been found that the behavior of heat transfer at the interface changes from heat loss to heat gain when the ambient temperature is increased. Moreover, the presence of partition block under normal gravity suppresses surface heat loss as well as surface heat gain similar to microgravity conditions. Streamlines and temperature contours have been presented for various conditions in order to clarify the underlying physics more meaningfully. The computed profiles for velocity and temperature at the liquid–air interface have been validated against established experimental results.     

Initial transient in Zn-doped InSb grown in microgravity

Three Zn-doped InSb crystals were directionally solidified under microgravity conditions at the International Space Station (ISS) Alpha. The distribution of the Zn was measured using SIMS. A short diffusion-controlled transient, typical for systems with k >1 was demonstrated. Static pressure of ∼4000 N/m² was imposed on the melt, to prevent bubble formation and de-wetting. Still, partial de-wetting has occurred in one experiment, and apparently has disturbed the diffusive transport of Zn in the melt.