Protein crystal growth in microgravity-temperature induced large scale crystallization of insulin

One of the major stumbling blocks that prevents rapid structure determination using x-ray crystallography is macromolecular crystal growth. There are many examples where crystallization takes longer than structure determination. In some cases, it is impossible to grow useful crystals on earth. Recent experiments conducted in conjunction with NASA on various Space Shuttle missions have demonstrated that protein crystals often grow larger and display better internal molecular order than their earth-grown counterparts. This paper reports results from three Shuttle flights using the Protein Crystallization Facility (PCF). The PCF hardware produced large, high-quality insulin crystals by using a temperature change as the sole means to affect protein solubility and thus, crystallization. The facility consists of cylinders/containers with volumes of 500, 200, 100, and 50 ml. Data from the three Shuttle flights demonstrated that larger, higher resolution crystals (as evidenced by x-ray diffraction data) were obtained from the microgravity experiments when compared to earth-grown crystals.     

Effect of convective disturbances induced by g-jitter on the periodic precipitation of lysozyme

Numerical simulations are carried out to investigate the crystallization process of a protein macromolecular substance under two different conditions: pure diffusive regime and microgravity conditions present on space laboratories. The configuration under investigation consists of a protein reactor and a salt chamber separated by an “interface”. The interface is strictly related to the presence of agarose gel in one of the two chambers. Sedimentation and convection under normal gravity conditions are prevented by the use of gel in the protein chamber (pure diffusive regime). Under microgravity conditions periodic time-dependent accelerations (g-jitter) are taken into account. Novel mathematical models are introduced to simulate the complex phenomena related to protein nucleation and further precipitation (or resolution) according to the concentration distribution and in particular to simulate the motion of the crystals due to g-jitter in the microgravity environment. The numerical results show that gellified lysozyme (crystals “locked” on the matrix of agarose gel) precipitates to produce “spaced deposits”. The crystal formation results modulated in time and in space (Liesegang patterns), due to the non-linear interplay among transport, crystal nucleation and growth. The propagation of the nucleation front is characterized by a wavelike behaviour. In microgravity conditions (without gel), g-jitter effects act modifying the phenomena with respect to the on ground gellified configuration. The role played by the direction of the applied sinusoidal acceleration with respect to the imposed concentration gradient (parallel or perpendicular) is investigated. It has a strong influence on the dynamic behaviour of the depletion zones and on the spatial distribution of the crystals. Accordingly the possibility to obtain better crystals for diffraction analyses is discussed.     

用于晶体生长的地基无容器悬浮技术

空间微重力环境下几乎无对流和沉降,可为晶体生长提供一个相对稳定和均一的理想环境,易于得到尺寸较大的高质量单晶。但是,空间结晶实验成功率低,费用昂贵,实验机会受限。因此,研发各种空间微重力环境地基模拟技术具有重要意义。目前可用于晶体生长的地基无容器悬浮技术主要有空气动力悬浮、静电悬浮、电磁悬浮、液体界面悬浮、超声悬浮和磁场悬浮技术等。这些地基模拟技术可实现晶体的无容器悬浮生长,避免器壁对晶体生长的不良影响,提高晶体质量,为解决X射线单晶衍射技术中的瓶颈问题提供新途径,还可为在地基进行结晶动力学和机理研究提供简单易行的方法。从技术原理、优势、缺陷及在结晶(特别是蛋白质结晶)中的应用4个方面对这些技术逐一进行了介绍和评述。重点介绍了液体界面悬浮、超声悬浮和磁场悬浮技术这3种用于蛋白质晶体生长的较为成熟的地基无容器悬浮技术。     

空间微重力环境下蛋白质晶体生长的研究进展

空间微重力环境可消除或减弱常重力场下溶液中存在的对流和沉降,为蛋白质晶体生长提供一个相对均一和稳定的环境,有利于得到尺寸更大、衍射分辨率更高的蛋白质晶体。通过对这些高质量空间晶体进行X射线衍射分析,可获得多种蛋白质的精细三维结构。从空间蛋白质晶体生长的发展历史、研究成果、生长机理、存在的问题与对策等方面总结了空间微重力环境下蛋白质晶体生长的研究进展,展望了空间蛋白质结晶的未来。     

JAXA-GCF project - high-quality protein crystals grown under microgravity environment for better understanding of protein structure

Since 2003, Japan Aerospace Exploration Agency (JAXA, former NASDA) has been conducting a project on a semi-annual basis (JAXA-GCF) to obtain high-quality protein crystals in the microgravity environment using the Russian transportation system. For this project, protein samples were mostly provided by Japanese users for whom JAXA provided technical and clerical support for crystallization experiments in microgravity. For the project, JAXA has constructed a user-friendly support service for microgravity experiments and provided regular and frequent flight opportunities. To simplify and improve technological matters, JAXA devised a gel-tube method crystallization device, which is effective both in space and on ground, based on the counter-diffusion technique. JAXA also provided ground-based techniques for efficient preliminary optimization of crystallization conditions using a 1-dimensional simulation and for harvesting and cryoprotecting crystals before X-ray diffraction experiments. These improvements have significantly increased the success rate of obtaining useful results. In conclusion, JAXA has developed technologies for growing, in microgravity, high-quality protein crystals, which may diffract up to atomic resolution, for a better understanding of 3-dimensional protein structures through X-ray diffraction experiments.     

Protein crystallization in space

The microgravity environment of space is an ideal place to study the complicated protein crystallization process and to grow good-quality protein crystals. A series of crystal growth experiments of 10 different proteins was carried out in space on a Chinese re-entry satellite FSW-2 in August, 1992. The experiments were performed for about two weeks at a temperature of 18.5 +/- 0.5 degrees C using a tube-like crystallization apparatus made in the Shanghai Institute of Technical Physics, Academia Sinica. More than half of 48 samples from 6 proteins produced crystals, and the effects of microgravity on protein crystal growth were observed, especially for hen-egg white lysozyme and an acidic phospholipase A2 from the venom of Agkistrodon halys Pallas. Analyses of the crystallization of these two enzymes in this mission showed that the microgravity environment in space may be beneficial to improve size, external perfection, morphology, internal order, and nucleation of protein crystals. Some of these positive microgravity effects were also demonstrated by the growth of protein crystals in gelled solution with the above two enzymes. A structural analysis of the tetragonal lysozyme crystal grown in space is in progress.     

Vapour phase crystal growth under microgravity environment

Vapour phase crystal growth experiments performed in the Skylab and ASTP missions are reviewed. The basic vapour phase crystal growth technique is described and effect of gravity is discussed. The multipurpose furnace specially designed to carry out various experiments in flight conditions is described. Ge Se, Ge Te and GeS as well as ternary GeSe_0·99 Te_0·01 and GeS_0·98 Se_0·02 crystals have been grown in space showing improvement over similarly grown crystals on ground as determined by x-ray diffraction, chomical homogeneity and surface morphology studies. Mass flux rates under microgravity conditions have been found to be up to 10 times larger than expected indicating need for better theoretical and experimental understanding of the effect of gravity on crystal growth.     

Sophisticated equipment developed for growth and evaluation of perfection of nearly perfect crystals

This paper reviews four major equipment developed at the National Physical Laboratory for growth and perfection evaluation of single crystals, namely (i) a crystal puller for growth of nearly perfect crystals by the Czochralski method; (ii) a microfocus x-ray generator; (iii) an x-ray diffraction topography camera; and (iv) a triple crystal x-ray diffractometer. The crystal puller can provide smooth, uniform and variable pulling rates. The maximum length of pull is nearly 60cm. Efforts have been made to isolate vibrations. Nearly perfect single crystals of KCl, KBr and NaCl with maximum diameter of ∼ 60 mm have been grown. The crystals give diffraction curves with half width in the range of 10–30 sec of arc. In the projection topographs, dislocations can be resolved and characterized. The microfocus x-ray generator is a demountable continuously evacuated system with specially designed electron gun and anode assembly. The vacuum is continuously monitored for ease of maintenance. In the point focus mode the spot size is 40 μm on the anode. X-ray topography system is a versatile equipment used for projection and section topography. It can provide 360° rotations to the specimen disc around an axis perpendicular to it. Rotations of a few sec of arc can be given to the specimen around a vertical axis. Typical diffraction curves of a dislocation-free crystal and a crystal with boundaries are shown. Well-resolved images of dislocations are shown in a topograph as an illustration. In the triple crystal x-ray diffractometer a highly collimated and monochromated Kα₁ exploring x-ray beam is obtained by combining microfocus source, a special collimator and crystal monochromators of Bonse-Hart type. With this beam very narrow diffraction curves with half width of about 5 sec of arc can be recorded. Typical results of measurement of diffuse x-ray scattering (dxs) on nearly perfect silicon single crystals are discussed. It has been observed that the contribution of phonons to thedxs is negligible. Thedxs is mainly due to point defect aggregates.     

Crystallization of ZSM-5 zeolite under microgravity

Crystal growth of ZSM-5 zeolite under microgravity was carried out using a Soviet reentry system. The space-grown zeolite was composed of grains of uniform shape and size (ca. 10 μm). Most of zeolite grains were linked with each other by the edge of the grain. The electron diffraction of each grain showed a single-crystal diffraction pattern, indicating the presence of well-crystallized zeolite crystal. The relatively large difference in the surface Al concentration existed between different crystals of the space-grown zeolite, as compared with the zeolite crystals synthesized in the ground with stirring. Based on these results, the crystals growth of zeolite in the microgravity environment was discussed.     

Does Microgravity Influence Self-Assembly??

The templated syntheses of TMA₂Sn3S7 and TBA2Sn4S9 (where TMA is tetramethylammonium and TBA is n-tetrabutylammonium) microporous layered tin(iv) sulfides have been carried out under both microgravity (μG) and earth (1G) conditions in order to elucidate the influence of gravity on the self-assembly and crystal-growth processes of this class of materials. The μG experiments were conducted on board the May 1996 Endeavour STS-77 NASA space-shuttle flight. It was determined that the long-range ordering of the porous layers and the population of defects but not the short-range ordering within the layers is influenced by gravity. Bulk and surface crystallinity, smoothness of crystal faces, optical quality, crystal habits, registry of the porous layers, and accessible void volume to adsorbates were found to be improved in the space-grown crystals. This is probably because the forces associated with the organization of the porous layers are expected to be weak and sensitive to the elimination of buoyancy-driven convective flows and Stokes sedimentation effects in a microgravity environment. One can draw an analogy to the weak forces between protein macromolecules and the established effect of microgravity on improving the diffraction quality of crystals harvested in space.     

微重力条件下熔体中对流过程的数值模拟

微重力条件下生长优质晶体遇到的最大问题是要控制晶体生长的条件，抑制由于重大的减弱而引起的熔体中的热毛细时流。但是，用实验来解决这些问题费用高，周期也长，而且有时完全用实验来模拟也是很困难的。用数值计算方法来模拟微重力条件下熔体中的对流过程是空间晶体生长研究的一个重要的方向，计算结果对控制空间生长晶体和抑制熔体中的对流有指导意义。对微重力条件下熔体中对流发生、发展的过程进行了数值研究。以有限差分法研究了沿上表面为自由表面的水平区域不同边界条件下的熔体中的对流过程。     

Thermomagnetic Convection as a Tool for Heat and Mass Transfer Control in Nanosize Materials Under Microgravity Conditions

Magnetic colloids are relatively new man-made nanomaterials whose magnetic susceptibility is several orders of magnitude larger than that of natural substances. Experiments conducted in magnetic fluids show that strengthening or weakening of thermal convection in colloids is dictated by a competition between the gravitational and magnetic mechanisms as well as by the effect of the fluid density stratification due to gravitational sedimentation of magnetic particles and their aggregates. Therefore experiments in microgravity conditions are required to eliminate gravitational sedimentation. This will enable an accurate investigation of convection in magnetic fluids and unambiguous study of the interaction of a magnetic field with a magnetopolarized medium. Such experiments are also needed to perform an accurate measurement of fluid’s transport coefficients.     

High Resolution Synchrotron X-Radiation Diffraction Imaging of Crystals Grown in Microgravity and Closely Related Terrestrial Crystals

Irregularities in three crystals grown in space and in four terrestrial crystals grown under otherwise comparable conditions have been observed in high resolution diffraction imaging. The images provide important new clues to the nature and origins of irregularities in each crystal. For two of the materials, mercuric iodide and lead tin telluride, more than one phase (an array of non diffracting inclusions) was observed in terrestrial samples; but the formation of these multiple phases appears to have been suppressed in directly comparable crystals grown in microgravity. The terrestrial seed crystal of triglycine sulfate displayed an unexpected layered structure, which propagated during directly comparable space growth. Terrestrial Bridgman regrowth of gallium arsenide revealed a mesoscopic structure substantially different from that of the original Czochralski material. A directly comparable crystal is to be grown shortly in space.     

Theory and simulation of buoyancy-driven convection around growing protein crystals in microgravity

We present an order-of-magnitude analysis of the Navier-Stokes equations in a time-dependent, incompressible and Boussinesq formulation. The hypothesis employed of two different length scales allows one to determine the different flow regimes on the basis of the geometrical and thermodynamical parameters alone, without solving the Navier-Stokes equations. The order-of-magnitude analysis is then applied to the field of protein crystallization, and to the flow field around a crystal, where the driving forces are solutal buoyancy-driven convection, from density dependence on species concentration, and sedimentation caused by the different densities of the crystal and the protein solution. The main result of this paper is to provide predictions of the conditions in which a crystal is growing in a convective regime, rather than in the ideal diffusive state, even under the typical microgravity conditions of space platforms.     

Theory and simulation of buoyancy-driven convection around growing protein crystals in microgravity

We present an order-of-magnitude analysis of the Navier-Stokes equations in a time-dependent, incompressible and Boussinesq formulation. The hypothesis employed of two different length scales allows one to determine the different flow regimes on the basis of the geometrical and thermodynamical parameters alone, without solving the Navier-Stokes equations. The order-of-magnitude analysis is then applied to the field of protein crystallization, and to the flow field around a crystal, where the driving forces are solutal buoyancy-driven convection, from density dependence on species concentration, and sedimentation caused by the different densities of the crystal and the protein solution. The main result of this paper is to provide predictions of the conditions in which a crystal is growing in a convective regime, rather than in the ideal diffusive state, even under the typical microgravity conditions of space platforms.     

A Compact Device for Colloidal Crystal Studies on Tiangong-1 Target Spacecraft

An experimental device with three crystallization cells, each with two working positions, was designed to study growth kinetics and structural transformation of colloidal crystals under microgravity condition. The device is capable of remote control of experimental procedures. It uses direct-space imaging with white light to monitor morphology of the crystals and reciprocal-space laser diffraction (Kossel lines) to reveal lattice structure. The device, intended for colloidal crystal growth kinetics and structural transformation on Tiangong-1 target spacecraft, had run on-orbit for more than one year till the end of the mission. Hundreds of images and diffraction patterns were collected via the on-ground data receiving station. The data showed that single crystalline samples were successfully grown on the orbit. Structural transformation was carefully studied under electric and thermal field. Using a backup device, control experiments were also performed on the ground under similar conditions except for the microgravity. Preliminary results indicated that the on-orbit crystals were more stable than the on-ground ones.     

The effect of gravity on freely growing crystals

The effect of surface tensions and gravity on freely growing crystals has been formulated in simple mathematical expressions. This gives an opportunity to predict the shape of a crystal, freely grown in space under microgravity as well as in a gravity field. Limitations for crystal growth in a gravity field are also given.     

The Biological Macromolecule Crystallization Database and NASA Protein Crystal Growth Archive

The NIST/NASA/CARB Biological Macromolecule Crystallization Database (BMCD), NIST Standard Reference Database 21, contains crystal data and crystallization conditions for biological macromolecules. The database entries include data abstracted from published crystallographic reports. Each entry consists of information describing the biological macromolecule crystallized and crystal data and the crystallization conditions for each crystal form. The BMCD serves as the NASA Protein Crystal Growth Archive in that it contains protocols and results of crystallization experiments undertaken in microgravity (space). These database entries report the results, whether successful or not, from NASA-sponsored protein crystal growth experiments in microgravity and from microgravity crystallization studies sponsored by other international organizations. The BMCD was designed as a tool to assist x-ray crystallographers in the development of protocols to crystallize biological macromolecules, those that have previously been crystallized, and those that have not been crystallized.     

Crystal growth from solutions under microgravity environment

The growth of large and perfect single crystals are now of considerable interest because of their increasing scientific and industrial importance. Any imperfection in the crystal causes malfunctioning, rapid aging, low reliability and low yield in manufacture. Due to the availability of earth orbiting spaceships, there is a possibility of growing these crystals with high purity. In this review paper, the author discusses mainly the growth of crystals under microgravity conditions (in spaceships) and their basic principles and also their relative merits.     

穗状晶形成及其结构的研究

凝固过程中的流体流动效应是现代凝固科学研究的热点,尤其是流动与凝固界面的耦合作用及流动对生长成的晶体质量的影响。作者自行设计了一套带有对流驱动系统的强制性晶体生长装置,可以实现液相层流作用下的晶体生长过程,而且流动的发生及流速的改变极为方便。利用透明模型合金 SCN-2%wt.Ace 作为研究对象,详细考察了液相快速流动作用下的强制性晶体生长的行为,首次发现了一种新的晶体形态——穗状晶;作者对其结构和形成过程进行了详细分析,并预示了这种结构的潜在应用价值。