The space experiment of protein crystallization aboard the Chinese spacecraft SZ-3

Using new flight hardware, a Chinese mission of space protein crystallization has been performed aboard the Chinese spacecraft SZ-3. Preliminary analyses of the experimental results have shown that a few proteins produced better crystals in space. At least, the crystals of cytochrome b5 mutant could diffract X-ray beyond the highest resolution reported so far for the same kind of crystals. In addition, some rules derived from our numerical studies of the liquid/liquid diffusion protein crystallization were proved by the crystallization of lysozyme as model protein in this space experiment, which also clearly showed the advantages and disadvantages of the gelation of the protein solution used in microgravity growth of protein crystals.     

Effects of a microgravity environment on the crystallization of biological macromolecules

Macromolecules crystals are indispensable intermediates in the analysis of macromolecular structure, are essential for the application of x-ray diffraction methods, and are at the same time the greatest obstacle to success. Protein crystals are generally difficult to grow, often of imperfect form or small size, and frequently lack sufficient order. Their growth has become the rate limiting step in x-ray crystallography. Evidence has emerged from protein crystallization experiments carried out in space that suggests macromolecular crystals of improved order and quality can be grown in a microgravity environment. Presumably the absence of density driven convection and sedimentation permits a more deliberate and graceful entry of individual molecules into the crystal lattice. This in turn results in improvements in both morphology and the diffraction patterns of the crystals. The precise mechanisms for these improvements and the means for their optimization are, however, not understood at more than a rudimentary level. I attempt here to review microgravity effects that may play a role in protein crystal growth, sedimentation, convection and surface contact, and suggest their possible mechanisms.     

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.     

Protein crystal growth in microgravity using a liquid/liquid diffusion method

Crystallization of proteins by liquid liquid diffusion method was performed in microgravity using the MDA Minilab aboard the US Space Shuttle. Three proteins, namely lysozyme, trichosanthin, and a new lechin, were crystallized in the space experiment. In contrast to the results of space experiments with a tube-like vapor diffusion method, the crystallization conditions for growing better crystals in space are remarkably different from the conditions optimized on earth. This may be due to difficulties in ground optimization, which are caused by gravity-dependent phenomena, in particular the specific convective flow occurring with liquid liquid diffusion.     

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.     

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。     

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.     

Counterdiffusion protein crystallisation in microgravity and its observation with PromISS (protein microscope for the international space station)

The crystallisation by counterdiffusion is a very efficient technique for obtaining high-quality protein crystals. A prerequisite for the use of counterdiffusion techniques is that mass transport must be controlled by diffusion alone. Sedimentation and convection can be avoided by either working in gelled systems, working in systems of small dimensions, or in the absence of gravity. We present the results from experiments performed on the ISS using the Protein Microscope for the International Space Station (PromISS), using digital holography to visualise crystal growth processes. We extensively characterised three model proteins for these experiments (cablys3*lysozyme, triose phosphate isomerase, and parvalbumin) and used these to assess the ISS as an environment for crystallisation by counterdiffusion. The possibility to visualise growth and movement of crystals in different types of experiments (capillary counterdiffusion and batch-type) is important, as movement of crystals is clearly not negligible.     

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.     

Experimental investigation of Marangoni convection and vibration-induced crystal motion during protein crystal growth

The occurrence of Marangoni convection during cytochrome c’ crystal growth and vibration-induced motion of lysozyme crystals were investigated using a High Density Protein Crystal Growth (HDPCG) apparatus. Particle image velocimetry was used to visualize fluid motion, but no particle motion was observed, which suggests that under the experimental conditions used, Marangoni convection is not a significant cause of fluid and crystal motion. When horizontal vibrations of controlled amplitude and frequency were applied to the HDPCG apparatus, lysozyme crystals located on the liquid-vapour interface of the HDPCG cell made significant movements up to 0.5mm in amplitude and velocities reaching 0.06mm/s. These results from the Marangoni convection and horizontal vibration experiments suggest that protein crystal movements observed in past space experiments were most likely caused by g-jitter on the spacecraft rather than Marangoni convection.     

Crystallization of the EGF receptor ectodomain on US space mission STS-47

Although biochemists working in the field of biological signal transduction have characterized cell surface receptors for numerous growth factors within the past ten years, none of the three-dimensional structures could be obtained for these important proteins which represent major components of the cells' growth control system. Now, the extracellular ligand binding domain of the EGF receptor was crystallized in the presence of EGF under microgravity on US Shuttle mission STS-47. In 8 out of 9 experiments prepared under different conditions crystal growth was observed. One of these space-grown crystals showed higher diffraction quality than all crystals previously obtained in the laboratory. It allowed, for the first time, evaluation of the real space group by partial data collection.     

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.     

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.     

Colloidal hard-sphere crystallization kinetics in microgravity and normal gravity

The hard-sphere disorder-order transition serves as the paradigm for crystallization. We used time-resolved Bragg light scattering from the close-packed planes to measure the kinetics of nucleation and growth of colloidal hard-sphere crystals. The effects of gravity are revealed by comparison of the experiments in microgravity and normal gravity. Crystallites grow faster and larger in microgravity, and the coarsening between crystallites is suppressed by gravity. The face-centered-cubic structure was strongly indicated as being the stable structure for hard-sphere crystals. For a sample with a volume fraction of 0.552, the classic nucleation and growth picture is followed.     

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.     

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.     

High-throughput screens for postgenomics: studies of protein crystallization using microsystems technology

This paper describes the fabrication of a micromachined miniaturized array of chambers in a 2-mm-thick single crystal (100) silicon substrate for the combinatorial screening of the conditions required for protein crystallization screening (including both temperature and the concentration of crystallization agent). The device was fabricated using standard photolithography techniques, reactive ion etching (RIE) and anisotropic silicon wet etching to produce an array of 10 x 10 microchambers, with each element having a volume of 5 microL. A custom-built temperature controller was used to drive two peltier elements in order to maintain a temperature gradient (between 12 and 40 degrees C) across the device. The performance of the microsystem was illustrated by studying the crystallization of a model protein, hen egg white lysozyme. The crystals obtained were studied using X-ray diffraction at room temperature and exhibited 1.78 A resolution. The problems of delivering a robust crystallization protocol, including issues of device fabrication, delivery of a reproducible temperature gradient, and overcoming evaporation are described.     

Effect of gravity on crystallization in heavy metal fluoride glasses processed on the T-33 parabolic flight aircraft

Heavy metal fluoride (HMF) glasses are prone to micro-crystal formation and resulting optical degradation during their high-temperature processing for fibre drawing. It is believed that the absence of gravity-driven density segregation in microgravity can reduce this undesired micro-crystallization during the processing of HMF glasses. Experiments were conducted on the T-33 parabolic flight aircraft in microgravity and under 2-g acceleration to study the effect of gravity on crystallization in HMF glasses. These preliminary experiments indicated that gravity enhances crystallization in HMF glasses during their processing at 370–400°C. However, these results were not considered conclusive due to the short duration of 20 seconds available on the parabolic flight aircraft. Subsequent ground-based experiments were conducted on the T-33 payload using statistical design of experiments to simultaneously study the effect of glass composition, processing temperature, processing time and mode of heating (continuous or pulsed). These experiments indicated that a continuous processing time of over two minutes at crystallization temperatures is required to observe a statistically significant amount of crystallization in HMF glasses. This established the need for longer duration experiments on the sounding rocket, space shuttle or space station.     

Kinetic Model of the Formation of Protein Crystals in Capillaries by Counterdiffusion

A kinetic model of crystallization of lysozyme is proposed, and computer calculations of this process are carried out. The conditions for the formation of such crystals are determined. Under these conditions, individual crystals were grown that were suitable for X-ray examination. The developed model enables prediction of the quantity, size, and place of crystal nucleation inside the capillary.