Japan Aerospace Exploration Agency Protein Crystallization Growth (JAXA PCG) - 02.03.17
The High Quality Protein Crystal Growth Experiment (JAXA PCG) is aimed at the growth of crystals of biological macromolecules by the counter-diffusion technique. The main scientific objective of the JAXA PCG experiment is to make high quality protein crystals in the microgravity environment. Science Results for Everyone
Protein crystallization experiments have been performed in space for more than 20 years. In this experiment, more than 300 protein samples are launched and high quality crystals successfully grown from about 80 percent of them. It is expected that a protein depletion zone and an impurity depletion zone are formed around a crystal during protein crystal growth if the process is not disturbed by gravity, thus giving better quality crystals. A new technique to estimate growth rate and impurity proves that, in microgravity, protein depletion and impurity depletion zones appear. Detailed analysis of high quality protein crystal structures is useful in designing new pharmaceuticals and catalysts for a wide range of industries. Experiment Details
OpNom: JAXA PCG
Kazunori Ohta, Japan Aerospace Exploration Agency (JAXA), Tsukuba, Japan
Mitsugu Yamada, Japan Aerospace Exploration Agency, Ibaraki, Japan
Japan Aerospace Exploration Agency (JAXA), Tsukuba, Japan
Sponsoring Space Agency
Japan Aerospace Exploration Agency (JAXA)
Japan Aerospace Exploration Agency
Earth Benefits, Scientific Discovery
ISS Expedition Duration
April 2009 - September 2010; March 2011 - May 2012; September 2012 - September 2013; March 2014 - March 2016; March 2016 - February 2017; March 2017 - September 2017; -
Inc20, 22/23, 24/25, 28, 30, 35, 39, 41, 44, 47, 49(TBD), 51(TBD)
- Under microgravity conditions, convection and sedimentation are suppressed. Therefore, diffusion areas are maintained, the density around the crystals decreases, the crystals grow slowly, the capture of impurities and microcrystal decreases, and a crystal will grow well.
- High quality protein crystals are obtained.
- Using high quality crystals obtained in the space experiment, detailed information on crystal structures for designing new drugs for diseases and catalysts for ecological application are obtained.
JAXA has developed a new technique that estimates the driving force ratio of crystals grown on the ground and in space, and the capture ratio of impurities through the diffusion/capture coefficient of protein. This technique proved that under microgravity conditions with high viscosity and slow diffusion, a protein depletion zone and impurity depletion zone appears. Using this technique we try to get high quality protein crystals. Our goal is to contribute to yielding results which meet the social requirements.
This experiment contributes to the complex process of drug discovery by revealing disease-related protein structure, and the production of new catalysts for the environmental and energy industries. This will show how effective the ISS is for investigations of this type.
This experiment contributes to the complex process of drug discovery by revealing disease-related protein structure, and the production of new catalysts for the environmental and energy industries.
Operational Requirements and Protocols
- After docking to ISS, move the PCG Canisters to JPM within 24 hours except in the event of 4-orbits docking. In the event of 4-orbits docking, whithin 35 hours.
- Protein Crystallization Research Facility (PCRF) controls the temperature at 20°C.
- After experiment, remove the PCG Canisters from PCRF cell tray and pack for return within 6 hours before Soyuz hatch closing. And retrieve on Soyuz.
- Loading the protein samples into crystallization cells in Russia (Moscow) and Baikonur.
- Install the crystallization cells into Crystallization Canister in Baikonur launch site.
- Launch the 2 sets of Canister on Soyuz/Progress.
- After docking to ISS, move the Canisters to JPM.
- Install the Canisters into the cell tray of Protein Crystallization Research Facility (PCRF) and start the experiment for around 7 weeks at 20°C.
- Crystal growth starts automatically, and the temperature control is started from ground control. No trigger is required to onboard crew.
- After experiment, remove the Canisters from PCRF cell tray and pack for return. Retrieve on Soyuz.
- Analyze the protein 3D structure using space grown crystals at the synchrotron facility (Ex, SPring-8, Photon Factory) on the ground.
Decadal Survey Recommendations
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The JAXA PCG studies have been performed for more than 20 years. JAXA has conducted protein crystallization experiments aboard the International Space Station (ISS) since 2003. In this experiment, over 300 protein samples were launched in order to obtain high-quality crystals in space. In the past PCG missions, 60%-70% of the proteins experimented were crystallized as single crystals. Excellent diffraction data used to determine the precise structure of proteins were obtained for several different proteins. Detailed structural analysis of those proteins are currently being conducted.
In microgravity, the incorporation of molecules into the crystal depends highly on diffusion. The molecules may be allocated in order and the incorporation of impurity may be suppressed. Consequently, the nature of this microgravity environment brings growth to the highly ordered crystals. It is assumed that the formation of a protein depletion zone (PDZ) and an impurity depletion zone (IDZ) around growing crystals under a microgravity environment is due to the suppression of a convection flow. The combination of the crystal size (R), the diffusion coefficiency of the protein molecule (D), and the kinetic coefficiency for the protein molecule (ß), Rß/D, could be an index of the extent of these depletion zones. Larger ‘Rß/D’ are favorable to maximize the effect of the microgravity environment. 'D' can be decreased by using a high-viscous reagent such as polyethylene glycol (PEG) 8,000 for crystallization solution, and 'ß' can be increased by using a highly purified protein sample for crystallization. Researchers are now able to estimate microgravity effects and optimize the crystallization condition prior to performing microgravity experiments by referring to this ß/D value.
Here are some results of the successful crystallization experiments in space.
The crystals of Aspergillus oryzae alpha-amylase were obtained as cluster-like crystals that diffracted up to 1.4 Å resolution on the ground. However, after further purification of the protein sample using FPLC, and changing the precipitant from salt to high viscous polyethylene glycol (PEG) 8,000, high-quality crystals were obtained. These crystals diffracted up to 0.79 Å resolution by visual inspection; and a full X-ray diffraction dataset could be obtained up to 0.92 Å resolution. After the data analysis, the electron density corresponding to hydrogen atoms were visualized.
Hematopoietic prostaglandin D synthase (H-PGDS) and Lipocalin-type prostaglandin D synthase (L-PGDS) are both clinically important drug target proteins provided by Professor Urade of University of Tsukuba.
H-PGDS was crystallized in space 12 times with more than 20 inhibitors since 1997. It was difficult to obtain good crystals of H-PGDS initially. Accordingly, scientists used the same strategy, which applied to the crystallization of alpha-amylase, to the crystallization of H-PGDS (using PEG as a high-viscous precipitant and highly purified protein sample). Later, investigators were able to obtain high-quality crystals of H-PGDS with new inhibitors that diffracted X-ray waves up to 1.1 Å. The inhibitors are expected to be candidates for new drug designs.
L-PGDS with a C65A mutation was previously crystallized with citrate or malonate as a precipitant, and the X-ray crystal structure was determined at 2.0 Å resolution. Scientists attempted to obtain high-quality crystals of the C64A mutant in a microgravity environment using the same conditions used in the previous study, but they did not obtain satisfactory results. Instead, they used the strategy mentioned above and were able to obtain high-quality crystals in microgravity, which diffracted at around 1.0 Å resolution. The crystal quality had clearly improved with the use of a high-viscosity precipitant solution and a highly purified protein in microgravity.
These examples are part of JAXA’s procedure for growing high-quality protein crystals. From the purification of a protein sample to the high-resolution X-ray data collection, including the optimization of crystallization conditions on the ground, JAXA has established a sequence of experimental steps for successful crystallization in space.
Safonova TN, Mordkovich NN, Polyakov KM, Manuvera VA, Veiko VP, Popov VO. Crystallization of uridine phosphorylase from Shewanella oneidensis MR-1 in the laboratory and under microgravity and preliminary X-ray diffraction analysis. Acta Crystallographica Section F: Structural Biology and Crystallization Communications. 2012 10/30/2012; 68(11): 1387-1389. DOI: 10.1107/S1744309112041784. PMID: 23143255.
Inaka K, Tanaka H, Takahashi S, Sano S, Sato M, Shirakawa M, Yoshimura Y. Numerical analysis of the diffusive field around a growing protein crystal in microgravity. Defect and Diffusion Forum. 2012 April; 323-325: 565-569. DOI: 10.4028/www.scientific.net/DDF.323-325.565.
Itoh T, Hibi T, Suzuki F, Sugimoto I, Fujiwara A, Inaka K, Tanaka H, Ohta K, Fujii Y, Taketo A, Kimoto H. Crystal structure of chitinase ChiW from Paenibacillus sp. str. FPU-7 reveals a novel type of bacterial cell-surface-expressed multi-modular enzyme machinery. PLOS ONE. 2016 December 1; 11(12): e0167310. DOI: 10.1371/journal.pone.0167310.
Yoshida H, Yoshihara A, Ishii T, Izumori K, Kamitori S. X-ray structures of the Pseudomonas cichorii D-tagatose 3-epimerase mutant form C66S recognizing deoxy sugars as substrates. Applied Microbiology and Biotechnology. 2016 July; epub: 13 pp. DOI: 10.1007/s00253-016-7673-7. PMID: 27368739.
Tanaka H, Tsurumura T, Aritake K, Furubayashi N, Takahashi S, Yamanaka M, Hirota E, Sano S, Sato M, Kobayashi T, Tanaka T, Inaka K, Urade Y. Improvement in the quality of hematopoietic prostaglandin D synthase crystals in a microgravity environment. Journal of Synchrotron Radiation. 2011 January 1; 18(1): 88-91. DOI: 10.1107/S0909049510037076.
Nakano H, Hosokawa A, Tagawa R, Inaka K, Ohta K, Nakatsu T, Kato H, Watanabe K. Crystallization and preliminary X-ray crystallographic analysis of Pz peptidase B from Geobacillus collagenovorans MO-1. Acta Crystallographica Section F: Structural Biology and Crystallization Communications. 2012; 68: 757-759. DOI: 10.1107/S1744309112018969.
Rahman RN, Ali MS, Leow TC, Salleh AB, Basri M, Matsumura H. The Effects of Microgravity on Thermostable T1 Lipase Protein Crystal. Gravitational and Space Biology. 2010; 23(2): 89-90.
Tanaka H, Inaka K, Furubayashi N, Yamanaka M, Takahashi S, Sano S, Sato M, Shirakawa M, Yoshimura Y. Controlling the diffusive field to grow a higher quality protein crystal in microgravity. Defect and Diffusion Forum. 2012 April; 323-325: 549-554. DOI: 10.4028/www.scientific.net/DDF.323-325.549.
Takahashi S, Tsurumura T, Aritake K, Furubayashi N, Sato M, Yamanaka M, Hirota E, Sano S, Kobayashi T, Tanaka T, Inaka K, Tanaka H, Urade Y. High-quality crystals of human haematopoietic prostaglandin D synthase with novel inhibitors. Acta Crystallographica Section F: Structural Biology and Crystallization Communications. 2010; 66(Pt. 7): 846-850. DOI: 10.1107/S1744309110020828.
Aris SN, Chor AL, Ali MS, Basri M, Salleh AB, Rahman RN. Crystallographic analysis of ground and space thermostable T1 lipase crystal obtained via counter diffusion method approach. BioMed Research International. 2014; 2014(904381): 8 pp. DOI: 10.1155/2014/904381.
Inaka K, Takahashi S, Aritake K, Tsurumura T, Furubayashi N, Yan B, Hirota E, Sano S, Sato M, Kobayashi T, Yoshimura Y, Tanaka H, Urade Y. High-Quality Protein Crystal Growth of Mouse Lipocalin-Type Prostaglandin D Synthase in Microgravity. Crystal Growth and Design. 2011 June; 11(6): 2107-2111. DOI: 10.1021/cg101370v.
Ground Based Results Publications
Timofeev VI, Smirnova E, Chupova L, Esipov RS, Kuranova IP. X-ray study of the conformational changes in the molecule of phosphopantetheine adenylyltransferase from Mycobacterium tuberculosis during the catalyzed reaction. Acta Crystallographica Section D: Biological Crystallography. 2012 11/09/2012; 68(12): 1660-1670. DOI: 10.1107/S0907444912040206.
Tanaka H, Sasaki S, Takahashi S, Inaka K, Wada Y, Yamada M, Ohta K, Miyoshi H, Kobayashi T, Kamigaichi S. Numerical model of protein crystal growth in a diffusive field such as the microgravity environment. Journal of Synchrotron Radiation. 2013 October 1; 20(6). DOI: 10.1107/S0909049513022784.
Takahashi S, Ohta K, Furubayashi N, Yan B, Koga M, Wada Y, Yamada M, Inaka K, Tanaka H, Miyoshi H, Kobayashi T, Kamigaichi S. JAXA Protein Crystallization in Space: Ongoing Improvements for Growing High-quality Crystals. Journal of Synchrotron Radiation. 2013 November; 20(6): 968-973. DOI: 10.1107/S0909049513021596.
High Quality Protein Crystallization Research (HQPC)
Protein Crystallization Research Facility. Image courtesy of JAXA.
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High Quality Protein Crystal Growth Experiment Canister. Image courtesy of JAXA.
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