This project aims to create a research initiative of high quality protein crystallization technology under a microgravity condition of the international space station (ISS). This technology has been developed in Japan through almost 20 years of effort and obtained many outstanding results, including development of orphan drug for patients with Duchenne muscular dystrophy, which is highly reputed from NASA and Boeing. The enzyme-utilizing and/or drug-developing companies in Japan strongly wish to use this technology, waiting for its maturation for applying to their product development. Although both the technology and the demand exist in this field, we have problem in a talent raising system. The technology was developed by individual labs and maintained within each lab, yet not shared with other labs. Therefore, a high barrier exists for young researchers to use, develop and diffuse the technology further. To solve these problems, we focused on three tasks in this program: (1) formulation of the technology platform, (2) technology transfer from senior to young researchers, and (3) building up the international and industrial network. In 2015 and 2016, we created a homepage of this research initiative (http://www.spaceprotein.com) and organized workshops for young researchers 7 times and international symposia and workshops twice. Through those activities, we have educated almost 150 young researchers for these 2 years and expanded the size of research initiative from the charter members of 6 to 30 and more at the end of 2016.

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High-quality crystals are expected to grow in microgravity environment. A protein depletion zone (PDZ) and an impurity depletion zone (IDZ) around a growing crystal are formed in microgravity environment because of the suppressions of convection flow. These depletion zones decrease concentrations of protein and impurities on the surface of the crystal, resulting in high quality crystal growth. Previously, we developed an easy method to estimate the effects of PDZ and IDZ in a steady state by using the combination of the diffusion coefficient and the kinetic constant of the protein molecule and the radius of the crystal, and have successfully applied it to increase microgravity effects on growing high-quality crystals. From this method, we learned that high-quality crystals could grow in microgravity if the crystals were larger, the protein sample was highly purified, and the crystallization solution was viscous. Recently, we introduced a numerical analysis of these depletion zones in a non-steady state and suggested that all the sections of the crystal are surrounded by different supersaturation levels of protein and different concentrations of impurity. Especially for the crystals grown on the ground, the concentration of impurity was higher in the center of the crystals. Our recent experimental results suggested that the cell dimensions of ground-grown crystals of lysozyme with some impurity were not uniform in a crystal. We speculated that it was because the disorder in the center of the crystal grown on the ground affected to the crystal lattice of the outer layer of the crystal. In this review, technical background of protein crystallization in space is reviewed.

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For successful protein crystallization in microgravity environment, the following four points are essential: high-quality protein samples and well-optimized crystallization conditions with high reproducibility, crystallization conditions which are optimized for counter-diffusion method, and crystallization conditions which enhance microgravity effects. To meet these points, we have a strategy that we first evaluate the protein samples and crystallization conditions by charge-density calculation, SDS-PAGE, Native-PAGE, high-performance ion-exchange column chromatography, DLS and preliminary crystallization experiment by vapor-diffusion method. Then according to the results of these evaluation, we make a trial of improvement of the protein samples and crystallization conditions. If the results of the evaluation are consistent with the charge density, good protein crystals grow in most cases. For the crystals grown in JAXA PCG (protein crystal growth) space experiment, harvesting and cryocooling of the crystals from capillaries require some skills. In this review, we introduce these techniques for users who have already participated or will participate in JAXA PCG.

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Protein X-ray crystallography is one of the most powerful methods to obtain the structural information at atomic resolution. However the crystallization is still most difficult process. And the diffraction resolution depends on the quality of crystals. Crystallization in microgravity environment is one of the most possible techniques for obtaining well-diffracted crystals. We have joined several space experiments of JAXA High-quality Protein Crystal Growth projects. These projects contain the crystallization in microgravity environment and improvements of techniques for high-resolution X-ray crystallography. Developments and improvements of the techniques are important to utilize the rare chance of experiments in microgravity environment. We report the modification of crystallization condition for microgravity environment and technical improvements for high-resolution X-ray crystallography from space grown crystals.

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X-ray structure analysis method has been widely utilized to solve the structure of biological macromolecules. It is, however, also well known that the hydrogen atoms and protons, which are important to understand the structure-function relationship of them, are hardly visible in the electron density map due to the small atomic scattering factor of hydrogen. To visualize hydrogen species, neutron structure analysis is the most powerful technique, because the nuclear scattering length of hydrogen is comparable to those of other atoms comprising the protein molecules. Though many x-ray crystal structures of [NiFe] hydrogenases were reported so far, the real proton pathway and reaction mechanism at the Ni-Fe active site are still unclear. In this paper, we report how to prepare the high-quality large single crystals by using an improved crystallization phase diagram for [NiFe] hydrogenase.

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Influenza A virus is a major human and animal pathogen with the potential to cause catastrophic loss of life. Influenza virus reproduces rapidly, mutates frequently, and occasionally crosses species barriers. The recent emergence of swine-origin influenza H1N1 and avian influenza related to highly pathogenic forms of the human virus has highlighted the urgent need for new effective treatments. Here, we describe two crystal structures of complexes made by fragments of PA and PB1, and PB1 and PB2. These novel interfaces are surprisingly small, yet they play a crucial role in regulating the 250 kDa. polymerase complex, and are completely conserved among swine, avian and human influenza viruses. Given their importance to viral replication and strict conservation, the PA/PB1 and PB1/PB2 interfaces appear to be promising targets for novel anti-influenza drugs of use against all strains of influenza A virus. It is hoped that the structures presented here will assist the search for such compounds.

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Water molecules play important roles in enzymatic reaction especially for hydrolases, which use water as a substrate. Neutron crystallography is a powerful tool for visualizing hydrogen atoms to understand protonation states of protein and orientation of water molecules, while it requires a large-volume crystal (>1 mm3 ) compared with X-ray crystallography. To overcome the hurdle of crystal volume, a counter diffusion device equipped with 2 mm diameter can be used at the microgravity experiments. We report here the crystallization of two cellulases which hydrolyse glycosidic linkages in cellulose.

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Some sugars or carbohydrates have been known to possess health-promoting functions and widely used as food additives. Glycoside Hydrolases (GHs) have been widely utilized for preparing those carbohydrate products but they basically catalyze cleavage or transfer of glycosidic bonds. Glycoside phosphorylases (GPs) and glycosynthases can efficiently catalyze elongation of glycosidic bonds and have potential for practical oligosaccharide synthesis. We have been studying structural basis of various carbohydrate-active enzymes, focusing on anomer-inverting GPs and glycosynthases. Here I review significance of the structural studies and discuss demand of high-quality protein crystallization technology in this research field.

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The purpose of “Alloy Semiconductor” experiment is to make clear the factors for crystal growth of a high-quality bulk alloy semiconductor by investigating (1) solute transport in liquid and (2) surface orientation dependence of growth kinetics under microgravity and terrestrial conditions. The temperature gradient furnace onboard “Kibo” is used for the growth of an InxGa1-xSb bulk crystal which is a potential substrate material of optoelectronic devices such as thermo-photo-voltaic cells and gas sensors, since the bang gap and the lattice constant of the crystals are tuned by adjusting the composition. The space experiment is briefly summarized in view of the past history from preparation for the flight.

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The manuscript reviews the microgravity experiments carried out onboard the International Space Station (ISS) to study the effects of gravity and orientation on the dissolution and growth properties of InGaSb ternary alloys. To study the effect of gravity, similar growth experiments were conducted under microgravity and normal gravity conditions. The effect of orientation was studied by the growth of InGaSb from (111)A and (111)B faces of GaSb (Ga and Sb faces) under microgravity onboard the ISS. The experimental results revealed that the growth rate was higher with better quality of crystal under microgravity than normal gravity. A model was proposed to explain the higher dissolution of GaSb (111)B than (111)A direction under microgravity. The higher growth rate of InGaSb from GaSb (111)B was found to be because of its higher dissolution of GaSb(111)B feed.

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We report the Hicari project summary, namely, history of the project, preparation of samples, preliminary experiments, determination of growth conditions, preparation for safety requirements, on orbit operations of the space experiments, results of the space grown SiGe crystals, lessons learned from the project and so on. In the series of experiments, the crystal growth parameters were adjusted by reflecting the results of the latest crystal growth experiment. We discuss two things; what is the best user integration process for the current JAXA materials science space experiments and the future work of the SiGe bulk crystal growth experiments.

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Total of four SiGe crystal growth experiments by the traveling liquidus-zone (TLZ) method have successfully been performed aboard the “Kibo” in 2013 and 2014. Results show that the TLZ method is a powerful method for growing compositionally uniform mixed crystals. On the ground, convection in a melt stops crystal growth and long crystals are difficult to be grown, while in microgravity long and large homogeneous crystals are grown in the diffusion limited regime. Step temperature change by 1 ℃ during crystal growth resulted in interface marking and growth rates in the axial and radial directions were measured precisely. Growth conditions for achieving radial uniformity were obtained. Growth instability at the initial stage was made clear, which was observed in microgravity for the first time. It is shown that convection in a melt has a merit of avoiding such instability.

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The foam production in space has been studied. The foam was generated by mixing surfactant-sugar solution with air in a porous media. The produced foam on the ground moved linearly, but it moved spirally under the microgravity. To explain the results, the foam production along the gravity was examined in a wide range of experimental conditions. In the foam motion state diagram dependent on air pump and liquid pump flow rates, the area of the spiral motion located at the higher air flow rate side and the area of the linear motion located at the lower air flow rate side. In higher surfactant concentrations the area of the spiral motion expanded by increasing the viscosity of the solution. On the other hand, in lower surfactant concentrations the area of the spiral motion shrank by increasing the viscosity of the solution.

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Foreign phase particles, which are engulfed by a growth front and then incorporated into a growing crystal, can cause a variety of problems. These problems can influence the crystal growth process and the preparation of the crystal or reduce the performance of resulting devices. In photovoltaics, the incorporation of SiC particles in VGF silicon leads to a relatively high material loss due to wire saw damages and shunts in the resulting solar cells. Due to the setup of the directional solidification the formation of SiC particles can hardly be avoided. Therefore, it is important to control the incorporation of the particles. It is known that the incorporation is dependent on the size of the particles and on the velocity of the moving solid-liquid interface. Existing theoretical models describe the transition between pushing and engulfment, but growth experiments show that the experimental values for the transition between these two states deviate significantly from the theoretical ones. In this work, several experiments under 1 G conditions and an experiment under µg conditions were done to investigate this question. The µg setup is necessary to get as close as possible to diffusive conditions which are the basic parameters for the theoretical calculations.

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Three Bridgman growth experiments were performed during the FOTON M4 mission in 2014. The aim of these experiments was to determine the influence of different convective states within the melt volume on the properties of the growing crystal. Transitions between different melt flow regimes were enforced during the ongoing growth by the application of rotating magnetic fields of different strengths and vibrations of different amplitude. A significant influence of the melt flow conditions on the Ga incorporation in the Ge crystal was observed for the µg crystals. The 1 G reference crystals, grown as well in the POLIZON 2 furnace (flight model) with the same parameters, showed always the expected dopant distribution as predicted by Scheil for complete mixing conditions. The buoyancy convection dominates the melt convection induced by the different RMFs or vibrations. The crystals were investigated by Differential Interference Contrast microscopy after structural etching, synchrotron topography to determine the structural quality, and spatially resolved resistivity measurements to determine the axial Ga segregation.

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This paper summarizes the panel discussion on the impact of microgravity on the diffraction properties of protein crystals. Convection-free environments have been considered as ideal environments for the growth of well diffracting protein crystals. Hence, since 1981 protein crystallization experiments have been performed in microgravity with varying degrees of success. During the discussion we addressed the effects of the crystal growth environment on crystal quality and perfection and we discussed whether higher crystal perfection leads to better X-ray diffractivity.

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