Sachiko Takahashi

A simplified counter diffusion method combined with a 1D simulation program for optimizing crystallization conditions

We developed a new protein crystallization method has been developed using a simplified counter-diffusion method for optimizing crystallization condition. It is composed of only a single capillary, the gel in the silicon tube and the screw-top test tube, which are readily available in the laboratory. The one capillary can continuously scan a wide range of crystallization conditions (combination of the concentrations of the precipitant and the protein) unless crystallization occurs, which means that it corresponds to many drops in the vapor-diffusion method. The amount of the precipitant and the protein solutions can be much less than in conventional methods. In this study, lysozyme and alpha-amylase were used as model proteins for demonstrating the efficiency of this method. In addition, one-dimensional (1-D) simulations of the crystal growth were performed based on the 1-D diffusion model. The optimized conditions can be applied to the initial crystallization conditions for both other counter-diffusion methods with the Granada Crystallization Box (GCB) and for the vapor-diffusion method after some modification.

High-quality crystals of human haematopoietic prostaglandin D synthase with novel inhibitors.

High-quality crystals of human haematopoietic prostaglandin D synthase in complex with novel inhibitors were obtained in microgravity.

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 PGH2 to PGD2 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 Å 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 Å 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.

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.

JAXA protein crystallization in space: ongoing improvements for growing high-quality crystals.

The Japan Aerospace Exploration Agency’s ‘high-quality protein crystal growth’ project is introduced. If crystallization conditions were carefully fixed in ground-based experiments, high-quality protein crystals grew in microgravity in many experiments on the International Space Station, especially when a highly homogeneous protein sample and a viscous crystallization solution were employed.

Improvement in the quality of hematopoietic prostaglandin D synthase crystals in a microgravity environment

Crystals of hematopoietic prostaglandin D synthase grown in microgravity show improved quality.

Diffusion coefficient of the protein in various crystallization solutions: The key to growing high-quality crystals in space

The diffusion coefficients of lysozyme and alpha-amylase were measured in the various polyethylene glycol (PEG) solutions. Obtained diffusion coefficients were studied with the viscosity coefficient of the solution. It was found that the diffusion process of the protein was suppressed with a factor of vγ, where ν is a relative viscosity coefficient of the PEG solution. The value of γ is −0.64 at PEG1500 for both proteins. The value increased to −0.48 at PEG8000 for lysozyme, while decreased to −0.72 for alpha-amylase. The equation of an approximate diffusion coefficient at certain PEG molecular weight and concentration was roughly obtained.

Numerical analysis of the depletion zone formation around a growing protein crystal.

Abstract: It is expected that a protein depletion zone and an impurity depletion zone are formed around a crystal during protein crystal growth if the diffusion field around the crystal is not disturbed. The growth rate of the crystal may be decreased and the impurity uptake may be suppressed to result in highly ordered crystals if these zones are not disturbed. It is well known that a microgravity environment can reduce convective fluid motion, and this is thought to disturb the depletion zones. Therefore, we expect that crystals grown in space can attain better quality than those grown on the ground. In this study, we estimate the depletion zone formation numerically and discuss the results of crystallization in space experiments. In case of α‐amylase, most of the crystals form a cluster‐like morphology on the ground using PEG 8000 as a precipitant. However, in space, we have obtained a single and high‐quality crystal grown from the same sample compositions. We have measured the viscosity of the solution, the diffusion coefficient, and the growth rate of protein crystals on the ground. Applying numerical analysis to these values a significant depletion zone was expected to form mainly due to higher values of the viscosity. This might be one of the main reasons for better quality single crystals grown in space, where the depletion zone is thought to remain undisturbed. For protein crystallization experiments, salts are widely used as a precipitant. However, in that case, reduced concentration depletion zone effects can be expected because of a low viscosity. Therefore, if it is possible to increase the viscosity of the protein solution by means of an additive, the depletion zone formation effect would be enhanced to provide a technique that would be especially effective in space.

Optimization of salt concentration in PEG-based crystallization solutions

Optimal salt concentration in a PEG-based crystallization solution is important for successful crystal growth and can be predicted prior to performing crystallization experiments.

Crystallization of the archaeal transcription termination factor NusA: a significant decrease in twinning under microgravity conditions

The transcription termination factor NusA from Aeropyrum pernix was crystallized using a counter-diffusion technique in both terrestrial and microgravity environments. Crystallization under microgravity conditions significantly reduced the twinning content (1.0%) compared with terrestrially grown crystals (18.3%) and improved the maximum resolution from 3.0 to 2.29 A, with similar unit-cell parameters. Based on a comparison of the crystal parameters, the effect of microgravity on protein crystallization is discussed.