Control of strain in subgrains of protein crystals by the introduction of grown-in dislocations.

It is important to reveal the exact cause of poor diffractivity in protein crystals in order to determine the accurate structure of protein molecules. It is shown that there is a large amount of local strain in subgrains of glucose isomerase crystals even though the overall crystal quality is rather high, as shown by clear equal-thickness fringes in X-ray topography. Thus, a large stress is exerted on the subgrains of protein crystals, which could significantly lower the resistance of the crystals to radiation damage. It is also demonstrated that this local strain can be reduced through the introduction of dislocations in the crystal. This suggests that the introduction of dislocations in protein crystals can be effective in enhancing the crystal quality of subgrains of protein crystals. By exploiting this effect, the radiation damage in subgrains could be decreased, leading to the collection of X-ray diffraction data sets with high diffractivity.

Oscillations and accelerations of ice crystal growth rates in microgravity in presence of antifreeze glycoprotein impurity in supercooled water.

The free growth of ice crystals in supercooled bulk water containing an impurity of glycoprotein, a bio-macromolecule that functions as 'antifreeze' in living organisms in a subzero environment, was observed under microgravity conditions on the International Space Station. We observed the acceleration and oscillation of the normal growth rates as a result of the interfacial adsorption of these protein molecules, which is a newly discovered impurity effect for crystal growth. As the convection caused by gravity may mitigate or modify this effect, secure observations of this effect were first made possible by continuous measurements of normal growth rates under long-term microgravity condition realized only in the spacecraft. Our findings will lead to a better understanding of a novel kinetic process for growth oscillation in relation to growth promotion due to the adsorption of protein molecules and will shed light on the role that crystal growth kinetics has in the onset of the mysterious antifreeze effect in living organisms, namely, how this protein may prevent fish freezing.

Correction of the equilibrium temperature caused by slight evaporation of water in protein crystal growth cells during long-term space experiments at International Space Station.

The normal growth rates of the {110} faces of tetragonal hen egg-white lysozyme crystals, R, were measured as a function of the supersaturation σ parameter using a reflection type interferometer under µG at the International Space Station (NanoStep Project). Since water slightly evaporated from in situ observation cells during a long-term space station experiment for several months, equilibrium temperature T(e) changed, and the actual σ, however, significantly increased mainly due to the increase in salt concentration C(s). To correct σ, the actual C(s) and protein concentration C(p), which correctly represent the measured T(e) value in space, were first calculated. Second, a new solubility curve with the corrected C(s) was plotted. Finally, the revised σ was obtained from the new solubility curve. This correction method successfully revealed that the 2.8% water was evaporated from the solution, leading to 2.8% increase in the C(s) and C(p) of the solution.

Growth rate measurements of lysozyme crystals under microgravity conditions by laser interferometry.

The growth rate vs. supersaturation of a lysozyme crystal was successfully measured in situ together with the crystal surface observation and the concentration measurements onboard the International Space Station. A Michelson-type interferometer and a Mach-Zehnder interferometer were, respectively, employed for real-time growth rate measurements and concentration field measurements. The hardware development, sample preparation, operation, and analysis methods are described.

Stable growth mechanisms of ice disk crystals in heavy water.

Ice crystal growth experiments in heavy water were carried out under microgravity to investigate the morphological transition from a disk crystal to a dendrite. Surprisingly, however, no transition was observed, namely, the disk crystal or dendrite maintained its shape throughout the experiments, unlike the results obtained on the ground. Therefore, we introduce a growth model to understand disk growth. The Gibbs-Thomson effect is taken into account as a stabilization mechanism. The model is numerically solved by varying both an interfacial tension of the prism plane and supercooling so that the final sizes of the crystals can become almost the same to determine the interfacial tension. The results are compared with the typical experimental ones and thus the interfacial tension is estimated to be 20 mJ/m(2). Next, the model is solved under two supercooling conditions by using the estimated interfacial tension to understand stable growth. Comparisons between the numerical and experimental results show that our model explains well the microgravity experiments. It is also found that the experimental setup has the capability of controlling temperature on the order of 1/100 K.

Measurements of growth rates of an ice crystal from supercooled heavy water under microgravity conditions: basal face growth rate and tip velocity of a dendrite.

The growth of single ice crystals from supercooled heavy water was studied under microgravity conditions in the Japanese Experiment Module ''KIBO'' of the International Space Station (ISS). The velocities of dendrite tips parallel to the a axis and the growth rates of basal faces parallel to the c axis were both analyzed under supercooling ranging from 0.03 to 2.0 K. The velocities of dendrite tips agree with the theory for larger amounts of supercooling when the growth on the basal faces are not zero. At very low supercooling there is no growth on the basal faces. With increasing supercooling the basal faces start to grow, the growth rate changing as a function of supercooling with a power law with an exponent of about 2, with the exponent approaching 1 as supercooling increases further. We interpret the growth on the basal faces as being controlled by two-dimensional nucleation under low supercooling, with a change in the growth kinetics to spiral growth with the aid of screw dislocations with increasing supercooling then to a linear growth law. We discuss the combined effect of tip velocity and basal face kinetics on pattern formation during the growth of ice.

Investigation of the protein pre-crystallization solution using analytical ultracentrifugation.

Analytical ultracentrifugation was used to study the crystal growth units in hen egg-white lysozyme pre-crystallization solution. Solutions containing various concentrations of lysozyme and NaCl in 50 mM sodium acetate buffer were used for experiments. The crystallization solution was ultracentrifuged using a mode where the sedimentation and diffusion are in equilibrium. The protein concentration gradient in the centrifugation cell was measured by light absorption and the molecular weight was calculated from the concentration gradient data. The results were analyzed assuming that the molecules have no interaction with each other. In all solutions except for 0.4 M NaCl, 30 mg ml(-1) lysozyme solution, it was shown that the molecular weight falls in the range 12,000-16,500 Da. In 0.4 M NaCl, 30 mg ml(-1) lysozyme solution no analysis was made because crystals appeared at the bottom of the cell after centrifugation. Since the calculated molecular weight of lysozyme monomer is 14,400 Da, it was concluded that the lysozyme molecule predominantly exists as a monomer in undersaturated and supersaturated solutions.

Scientific approach to the optimization of protein crystallization conditions for microgravity experiments.

The National Space Development Agency of Japan (NASDA) developed a practical protocol to optimize protein crystallization conditions for microgravity experiments. This protocol focuses on the vapor diffusion method using high density protein crystal growth (HDPCG)--hardware developed by the University of Alabama, Birmingham--that flew on the STS-107 mission. The objective of this development was to increase the success rate of microgravity experiments by setting crystallization conditions based on knowledge of crystal growth and fluid dynamics. The protocol consists of four steps: (1) phase diagram preparation, (2) estimation of condensation rate in the vapor diffusion method, (3) fluid dynamic property measurement, and (4) fluid dynamic simulation. First, a phase diagram was constructed. Crystallization characteristics were investigated by a microbatch method. The data were recalculated based on classical nucleation theory and the crystallization boundary was determined as a function of time. The second step was to develop a practical model to estimate the condensation rate of the crystallizing solution, including protein and precipitant, as a function of the precipitant concentration and solution volume. By considering the crystallization map and the vapor diffusion condensation model we were able to optimize the crystallization conditions that generate crystals in the desired time. This was particularly important in a shuttle mission whose mission duration is limited. The third step was fluid dynamic property measurement necessary for fluid dynamics simulation and crystal growth study. The last step was to estimate the mass transport in space on the basis of the fluid dynamics simulation transport model. It turned out that neither the vapor phase nor the solution phase was seriously affected by gravity until nucleation provided the hardware was set in a normal direction. Therefore, we concluded that the optimized crystallization conditions could be directly applied to microgravity experiments. By completing the approach, we were able to control the time for nucleation in the vapor diffusion method.

Impurity effects on lysozyme crystal growth.

Atomic force microscopy (AFM) and X-ray diffraction experiments have been combined to study the correlation between impurity incorporation, crystal surface morphology and crystal quality. Hen egg-white lysozyme has been used as a model protein, and covalently bound lysozyme dimer as a model impurity. AFM observation of the [101] crystal face revealed that the crystal surface clearly became rough when 5% impurity was added, and the steps disappeared as the impurity concentration increased to 10%. The crystal quality was evaluated by four factors: maximum resolution limit, /, Rmerge, and overall B factor. In every index, the crystal quality tended to degrade as the impurity concentration increased. The B-factor dropped significantly at 5% impurity; at the same time the step roughening was observed. This strongly suggested that the impurity incorporation affected the step growth mechanism and degraded the crystal quality.

Improvement of protein crystal quality by forced flow solution.

Flow experiments in growing protein crystals were conducted to clarify the influence of the solution flow on the crystal quality. Lysozyme crystals grown under various flow velocities were analyzed by using synchrotron radiation to assess the quality. As a result, the crystals grown under forced flow were of better quality than those grown in quiescent conditions.