In-situ Observation of Growth Mechanisms of Protein Crystals and Their Perfection Under Microgravity (Nano Step) - 04.28.16
The In-situ Observation of Growth Mechanisms of Protein Crystals and their Perfection under Microgravity (NanoStep) investigation aims to clarify the relationship between crystal growth mechanism, surface morphology, and the perfection of crystals. Crystallization of proteins in microgravity yields crystals with better perfection than crystallization on Earth. The reason for this phenomenon has not been explained from a viewpoint of crystal growth mechanism. Science Results for Everyone
Laser interferometers have for the fist time been employed in ISS to measure the growth rate of lysozyme crystals versus the driving force, supersaturation of solution to investigate the difference of crystal growth mechanisms in gravity and under microgravity. The NanoStep experiment revealed that space grown lysozyme crystals grow faster (!) than the Earth-grown crystals by 30-50%. This result completely conflicts with a preexisting assumption; “space-grown crystals are better because they grow slower under convection-free environment.” This faster growth in space was kinetically analyzed due self-purification of impurities during growth coupled with a reduction of an impurity mediated crystal growth mechanism. Experiment Details
OpNom: Nano Step
Katsuo Tsukamoto, Tohoku University, Japan
Etsuro Yokoyama, Gakushuin University, Tokyo, Japan
Hajime Sasaki, Hokkaido University, Japan
Masaru Tachibana, Yokohama City University, Japan
Tetsuo Okutsu, Gunma University, Japan
Kenji Izumi, Yamaguchi University, Japan
Yoshinao Suzuki, Tokushima University, Japan
Izumi Yoshizaki, Japan Aerospace Exploration Agency, Tsukuba, Japan
Kenta Murayama, Tohoku University, Sendai, Japan
Sponsoring Space Agency
Japan Aerospace Exploration Agency (JAXA)
Japan Aerospace Exploration Agency
ISS Expedition Duration 1
May 2012 - March 2013
Previous ISS Missions
- It has been emphasized in the literature that the protein crystal perfection increases in microgravity conditions. However, the precise mechanism responsible for such observation is yet to be understood from a fundamental point of view.
- The objective is to study the correlation of growth mechanisms at various levels of supersaturation and defects induced during growth.
Lysozyme is used as a model protein and the growth of the crystal surfaces is observed using a Michelson interferometer.
This experiment will increase the efficiency of PCG experiments in microgravity.
The understanding of crystal growth mechanisms will increase the perfection of various crystals and benefit the people on earth.
Operational Requirements and Protocols
There are 3 samples with different concentrations. The experiment duration for one sample is 35 days. The crystal growth will be observed realtime by Michelson interferometry. The sample will not return.
Install the NanoStep Cell to SCOF, Ryutai Rack. When one sample observation is finished, then the sample will be exchanged by the crew member.^ back to top
Decadal Survey Recommendations
Information Pending^ back to top
20% of space-grown protein crystals were reported to be better in resolution in X-ray diffraction. However in recent years this “idea” has been claimed not always to be true because the Earth-grown crystals show sometimes better X-ray diffraction if the crystals were grown in gel, aqueous solution under a special configuration or in a flow. This discrepancy comes from the fact that so many protein crystals have been grown in space, but there have been no crucial experimental results to be compared in the difference of growth mechanism from a kinetic point of view. This means that the conventional way of investigation, namely, crystals grow in space and back to the ground for X-ray examination, is not sufficient. We therefore aimed to study the growth mechanism in space in more direct way by using in-situ interferometry for the NanoStep project. To achieve this, we measured the growth rate vs supersaturation and observe the surface topography of growing crystals. These two methodologies are known to be suitable for the analysis of crystal growth mechanism but interferometric investigation to get nano-scale in-situ information on crystal growth in space is not easy. We therefore developed an in-situ observation system coupling two laser interferometers suitable for ISS experiments. The new system successfully worked to finish all planned experiments in due period, August to December of 2013. This was the first time that the technique had been used onboard the ISS to measure the growth rate of the crystals at various temperatures with the same or even better precision as in gravity.
The researchers used two interferometers, Michelson type and Mach-Zehnder type interferometry for precise measurements of the growth rate of the lysozyme crystals vs their driving force, supersaturation and of protein concentration at the crystal surface, respectively. This yielded crucial information about the growth mechanism. The researchers remotely varied the supersaturation of the solution by increasing or decreasing the solution temperature. This took place over a range of 10 - 40 degrees Celsius, which necessitated building a closed growth cell to withstand the stresses caused by the thermal expansion of the growth solution and the growth cell. To extract true growth rate of the crystal face with an accuracy of 0.01nm/s from the expanding growth cell, three tiny reference mirrors were used in each growth cells. Three different cells have been prepared containing different dimer and protein concentrations. The preliminary results can be summarized as follows:
1. Growth rates of the crystal under microgravity are expected to be slower because of the suppression of solution convection, the results instead showed an increased growth rate. In order to get this new result, precise protein concentration by interferometry had to be measured onboard before and after the experiment because concentration of the solution gradually increased due to slow evaporation of water, ~1.5%, from the solution in this long term experiment. The growth rate was measured from a few spiral hillocks on the (110) face by carefully choosing best hillocks whose spirals steps do not interfere to the neighboring spiral hillocks. These were the keys for the success of this growth rate experiment.
2. The best-purified solution still shows a dead zone (no growth regime) at low supersaturation in space, though the width of the zone in space is smaller. In all experiments in ISS the effect of dimer impurities on the growth rate was less than that of the Earth-grown crystals. This is interpreted due to the slower diffusion rate of larger-sized impurities. This can be called to be a “self-purification” effect. However this effect could reasonably be expected not to be operated for smaller impurities in size.
3. Growth mechanisms predicted from the growth rate vs supersaturation are essentially not different from that in gravity. Spiral growth operates at low supersaturation followed by 2D nucleation growth with increasing supersaturation. However the regime of 2D heterogeneous nucleation growth that is mediated by the presence of dimer impurities on the surface is narrower. This may explain the better quality space-grown crystals because suppression of impurity mediated 2D heterogeneous nucleation growth would reduce the formation of micro-defects in the crystal lattices, which eventually leads to “better” quality of crystals. However it is questionable if the good quality or perfect crystals are the same as crystals which strongly diffracts X-ray, about which experiments are on-going using x-ray diffraction and X-ray topography, although perfect crystals are believed to diffract X-ray more strongly.
4. Microgravity condition is suitable for growing good lysozyme crystals in a wide range of supersaturation because of the lack of impurity mediated heterogeneous 2D nucleation growth. However we have to note that the same quality of crystals could be grown in gravity if we could extract nominal growth condition though space experiments. The spiral growth regime is ideal for the growth of good crystals both in gravity and in space. The spiral growth regime, however, in space was found to be much wider than in gravity.^ back to top
Fujiwara T, Suzuki Y, Yoshizaki I, Tsukamoto K, Murayama K, Fukuyama S, Hosokawa K, Oshi K, Ito D, Yamazaki T, Tachibana M, Miura H. 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. Review of Scientific Instruments. 2015 August; 86(8): 083704. DOI: 10.1063/1.4928491.
Suzuki Y, Tsukamoto K, Yoshizaki I, Miura H, Fujiwara T. First direct observation of impurity effects on the growth rate of tetragonal lysozyme crystals under microgravity as measured by interferometry. Crystal Growth and Design. 2015 August 31; 15(10): 4787-4794. DOI: 10.1021/acs.cgd.5b00456.
Ground Based Results Publications
Yoshizaki I, Tsukamoto K, Yamazaki T, Murayama K, Oshi K, Fukuyama S, Shimaoka T, Suzuki Y, Tachibana M. Growth rate measurements of lysozyme crystals under microgravity conditions by laser interferometry. Review of Scientific Instruments. 2013; 84(10): 103707. DOI: 10.1063/1.4826090. PMID: 24182119.
Yamazaki T, Tsukamoto K, Yoshizaki I, Fukuyama S, Miura H, Shimaoka T, Maki T, Oshi K, Kimura Y. Development of compartment for studies on the growth of protein crystals in space. Review of Scientific Instruments. 2016 March 1; 87(3): 033107. DOI: 10.1063/1.4942961.
NASA Image: ISS033E007364 - Close-up view of the Nano Step payload Specimen Cell prior to insertion into the SCOF (Solution Crystallization Observation Facility) in the Japanese Experiment Module (JEM).
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NASA Image: ISS033E007362 Japan Aerospace Exploration Agency astronaut Aki Hoshide,Expedition 33 flight engineer,services the Nano Step payload in the Japanese Experiment Module (JEM).
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NASA Image: ISS033E007383 - Close-up view of the Nano Step payload Specimen Cell to be located in the SCOF (Solution Crystallization Observation Facility).
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