Japan Aerospace and Exploration Agency - Granada Crystallization Facility High Quality Protein Crystallization Project (JAXA-GCF) - 07.15.14
ISS Science for Everyone
Science Objectives for Everyone In microgravity, JAXA-GCF grew high-quality crystals which are to be used in ground-based research to understand the structure of proteins within the human body.
Science Results for Everyone
This investigation sought to provide a low-cost, simple way to make high quality protein crystals in microgravity, using the counter-diffusion method. With the human genome completely decoded, research has moved to identifying the structures and functions of proteins based on genes, and large, well-ordered crystals are needed. It has been shown that cystals grown in microgravity are larger and more organized than those grown on Earth. Crystals grown on the space station could provide detailed knowledge of many important protein molecules used in pharmaceutical research for cancer treatments, stroke prevention, and other diseases.
European Space Agency (ESA), Noordwijk, , Netherlands
Granada University, Granada, , Spain
Sponsoring Space Agency
Japan Aerospace Exploration Agency (JAXA)
ISS Expedition Duration
April 2003 - October 2008
Previous ISS Missions
- The primary objective of JAXA-GCF was to provide a low-cost, simple experiment platform for the production of protein crystals in the microgravity environment.
- This experiment utilized the counter-diffusion method in space to grow high-quality protein crystals for x-ray diffraction studies of protein structure on Earth.
In April 2003, it was announced that the human genome had been completely decoded. Research has now moved to the next step, post-genome research, which involves identifying the structures and functions of proteins, based on genes. In order to learn more about protein functions, it is important to clarify the three-dimensional protein structure at the atomic level. In most cases, the structure of a protein is closely related to its function, so in-depth analysis of the three-dimensional protein structure is being linked to the study of protein functions.
Human life is supported by nearly 100,000 different kinds of proteins, most of whose structures are still unknown. By analyzing the structures of many different kinds of proteins and understanding how they interact with one another, it may become possible to identify various mechanisms involved in life phenomena. It should also become possible to inhibit or activate functions of certain proteins to develop new pharmaceuticals.
The JAXA-GCF investigation was a unique cooperation between several International Partners. The GCF hardware was originally developed at the University of Granada and utilized by the European Space Agency during Increments 3 and 5. For JAXA-GCF, the original GCF hardware was utilized and launched to ISS on a Russian vehicle with the cooperation of ESA and temperature was maintained using the Commercial Generic Bioprocessing Apparatus (CGBA) hardware in the U.S. Laboratory.
For the first, second and third space experiments, JAXA launched proteins of alpha-amylase and lysozyme to validate the counter-diffusion crystallization technique. As a result, high-quality protein crystals were successfully obtained. The final three experiments grew various crystals for post-genome research as well as pharmaceutical purposes. Some of the proteins grown include: Lipocalin - type Prostaglandin D Synthase 2 (L-PGDS) and Hematopoietic Prostaglandin D Synthase 2 (H-PGDS).
The GCF is a metallic box that houses 23 Granada Crystallization Boxes? (GCB) carefully conditioned in individual plastic bags and wrapped in a high absorbency cloth, and a temperature data logger. The GCF is a completely passive instrument that does not require any activation or crew intervention beyond installing it in the ISS where it is not exposed to inadvertent manipulations. It does not require any external supply. Proteins are crystallized in space inside an incubator on board the ISS.
The crystals grown in microgravity are able to grow larger and more organized than those grown on Earth. The results from this investigation may further human space exploration efforts by creating technological and biological advancements.
Biotechnology and pharmaceutical researchers carry out the process of protein crystallization in order to grow large, well-ordered crystals for use in x-ray diffraction studies. However, on Earth, the protein crystallization process is hindered by forces of sedimentation and convection since the molecules in the crystal solution are not of uniform size and weight. This leads to many crystals of irregular shape and small size that are unusable for x-ray diffraction. X-ray diffraction is a complex process which requires several months to several years to complete, and the quality of information obtained about the three-dimensional structure of a protein is directly dependent on the degree of perfection of the crystals. Thus, the structures of many important proteins remain a mystery simply because researchers are unable to obtain crystals of high quality or large size. Consequently, the growth of high quality macromolecular crystals for diffraction analysis has been of primary importance for protein engineers, biochemists, and pharmacologists.
Fortunately, the microgravity environment aboard the ISS is relatively free from the effects of sedimentation and convection and provides an exceptional environment for crystal growth. Crystals grown in microgravity could help scientists gain detailed knowledge of the atomic, three-dimensional structure of many important protein molecules used in pharmaceutical research for cancer treatments, stroke prevention and other diseases. The knowledge gained could be instrumental in the design and testing of new drugs.
Each Granada Crystallization Box (GCB) accommodates up to six capillaries with a maximum diameter of 1 mm to make up the Granada Crystallization Facility (GCF). The external dimensions of a GCB are 36x101x7 mm. The total weight of one GCB filled with six capillaries and the chemical solutions is 24 grams. No moving parts, or electrically-powered system are required to activate the experiments.
Preflight, a layer of buffer solution is formed at the bottom of the GCB reservoir and gelled. The capillaries, entirely filled with the protein solution, are then inserted through the holes of the guide and pressed to a given depth into the gelled buffer layer. Finally, the salt solution is poured onto the buffer layer and the box is hermetically sealed. The actual time at which the crystallization experiments start is determined by the thickness of gelled buffer that the precipitant has to diffuse through before it reaches the protein solution. The fact that the buffer is gelled makes this lag time insensitive to either manipulation or launch accelerations or vibrations. This lag time can be tuned to up to 3-4 days.
After launch, typically two days are required for the Soyuz spacecraft to reach the orbit of the International Space Station and dock to it. The GCF is extracted from its Soyuz transport container, transferred as soon as possible to ISS and stored in a quiet location (with stable microgravity and temperature (20 degrees C), and no vibrations), not to be touched by the crew anymore until the end of the mission. When samples are returned to the ground by Soyuz, insulators wrapped around the GCF form a thermos-like container and isolate the crystallization inside GCBs from fluctuations of ambient temperature.
The GCF is extracted from its Soyuz transport container as soon as possible after docking. It is transferred to an environmentally quiet location onboard the ISS where it will not be accidentally manipulated by the crew. The crew will not touch the GCF again until, at the end of the mission, it is transferred into the returning Soyuz.
Through the first, second and third space experiments, JAXA launched proteins of alpha-amylase and lysozyme to validate the developed crystallization technique. As a result, high-quality protein crystals were successfully obtained, and the structure was analyzed closely and carefully.
In some of the proteins (e.g., sleeping and allergy material synthetase, and proteins related to symptoms of parasite infection) provided by user organizations, JAXA also obtained the highest resolution data from the protein crystals grown in space. This was expected and can lead to new pharmaceutical development in the future. JAXA-GCF results improved maximum resolution in the range of 1 to 1.5 Angstrom (Kinoshita 2005).
Tanaka H, Inaka K, Inaka K, Sugiyama S, Takahashi S, Sano S, Sato M, Yoshitomi S. Numerical Analysis of teh Depletion Zone Formation Around a Growing Protein Crystal. Annals of the New York Academy of Sciences. 2004; 1027: 10-19.
Takahashi S, Tsurumura T, Aritake K, Furubayashi N, Sato M, Yamanaka M, Hirota E, Sano S, Kobayashi T, Tanaka T, Inaka K, 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.
Meyer A, Rypniewski W, Szymański M, Voelter W, Barciszewski J, Betzel C. Structure of mistletoe lectin I from Viscum album in complex with the phytohormone zeatin. Biochimica et Biophysica Acta. 2008; 1784(11): 1590-1595. DOI: 10.1016/j.bbapap.2008.07.010.
Tanaka H, Tsurumura T, Aritake K, Furubayashi N, Takahashi S, Yamanaka M, Hirota E, Sano S, Sato M, Kobayashi T, Tanaka T, Inaka K, Inaka K, Urade Y. Improvement in the quality of hematopoietic prostaglandin D synthase crystals in a microgravity environment. Journal of Synchrotron Radiation. 2010; 18: 88-91. DOI: 10.1107/S0909049510037076.
Timofeev V, Abramchik YA, Zhukhlistova N, Muravieva T, Fateev I, Esipov R, Kuranova IP. 3′-Azidothymidine in the active site of Escherichia coli thymidine phosphorylase: the peculiarity of the binding on the basis of X-ray study. Acta Crystallographica Section D: Biological Crystallography. 2014 April 1; 70(4): 11 pp. DOI: 10.1107/S1399004714001904.
Higashiura A, Kurakane T, Matsuda M, Suzuki M, Kobayashi T, Tanaka T, Tanaka H, Fujiwara T, Nakagawa A. High-resolution X-ray crystal structure of bovine H-protein at 0.88 Å resolution. Acta Crystallographica Section D: Biological Crystallography. 2010; 66: 698-708. DOI: 10.1107/S0907444910010668.
Kitatani T, Nakamura Y, Wada K, Kinoshita T, Tamoi M, Shigeoka S, Tada T. Structure of apo-glyceraldehyde-3-phosphate dehydrogenase from Synechococcus PCC7942. Acta Crystallographica Section F: Structural Biology and Crystallization Communications. 2006; 62: 727-730. DOI: 10.1107/S1744309106027916.
Yamanaka M, Inaka K, Inaka K, Furubayashi N, Matsushima M, Takahashi S, Tanaka H, Sano S, Sato M, Kobayashi T, Tanaka T. Optimization of salt concentration in PEG-based crystallization solutions. Journal of Synchrotron Radiation. 2011; 18: 84-87. DOI: 10.1107/S0909049510035995.
Kamauchi S, Urade Y. Hematopoietic prostaglandin D synthase inhibitors for the treatment of duchenne muscular dystrophy. Brain and Nerve. 2011; 63(11): 1261-1269. PMID: 22068479. [Japanese]
Sato M, Tanaka H, Inaka K, Inaka K, Shinozaki S, Yamanaka A, Takahashi S, Yamanaka M, Hirota E, Sugiyama S, Kato M, Saito C, Sano S, Motohara M, Nakamura T, Kobayashi T, Yoshitomi S, Tanaka T. JAXA-GCF project - high-quality protein crystals grown under microgravity environment for better understanding of protein structure. Microgravity Science and Technology. 2006 September; 18(3/4): 184-189. DOI: 10.1007/BF02870406.
Kuranova IP, Smirnova E, Abramchik YA, Chupova L, Esipov R, Akparov VK, Timofeev V, Kovalchuk VI. Crystal Growth of Phosphopantetheine Adenylyltransferase, Carboxypeptidase T, and Thymidine Phosphorylase on the International Space Station by the Capillary Counter-diffusion Method. Crystallography Reports. 2011 Sep; 56(5): 884-891. DOI: 10.1134/S1063774511050154.
Tanaka H, Yoshizaki I, Takahashi S, Yamanaka M, Fukuyama S, Sato M, Sano S, Motohara M, Kobayashi T, Yoshitomi S, Tanaka T. Diffusion Coefficient of the Protein in Various Crystallization Solutions: The Key to Growing High-quality Crystals in Space. Microgravity Science and Technology. 2006; 18(3/4): 91-94.
Tanaka H, Umehara T, Inaka K, Inaka K, Takahashi S, Shibata R, Bessho Y, Sato M, Sugiyama S, Fusatomi E, Terada T, Shirouzu M, Sano S, Motohara M, Kobayashi T, Tanaka T, Tanaka A, Yokoyama S. Crystallization of the archaeal transcription termination factor NusA: a significant decrease in twinning under microgravity conditions. Acta Crystallographica Section F: Structural Biology and Crystallization Communications. 2007 01/17/2007; 63(2): 69-73. DOI: 10.1107/S1744309106054625.
Kinoshita T, Maruki R, Warizaya M, Nakajima H, Nishimura S. Structure of a high-resolution crystal form of human trisephosphate isomerase: improvement of crystals using the gel tube method. Acta Crystallographica Section F: Structural Biology and Crystallization Communications. 2005; 61: 346-349. DOI: 10.1107/S1744309105008341.
Malecki PH, Rypniewski W, Szymański M, Barciszewski J, Meyer A. Binding of the plant hormone kinetin in th active site of Mistletoe Lectin I from Viscum album. Biochimica et Biophysica Acta. 2012; 1824: 334-338. DOI: 10.1016/j.bbapap.2011.10.013.
Ground Based Results Publications
Oda K, Matoba Y, Noda M, Kumagai T, Sugiyama M. Catalytic Mechanism of Bleomycin N-Acetyltransferase Proposed on the Basis of Its Crystal Structure. Journal of Biological Chemistry. 2009 11/03/2009; 285(2): 1446-1456. DOI: 10.1074/jbc.M109.022277.
Aritake K, Kado Y, Inoue T, Miyano M, Urade Y. Structural and Functional Characterization of HQL-79, an Orally Selective Inhibitor of Human Hematopoietic Prostaglandin D Synthase. Journal of Biological Chemistry. 2006; 281(22): 15277-15286. DOI: 10.1074/jbc.M506431200.
Mohri I, Taniike M, Taniguchi H, Kanekiyo T, Aritake K, Inui T, Fukumoto N, Eguchi N, Kushi A, Sasai H, Kanaoka Y, Ozono K, Narumiya S, Suzuki K, Urade Y. Prostaglandin D2-Mediated Microglia/Astrocyte Interaction Enhances Astrogliosis and Demyelination in twitcher. Journal of Neuroscience. 2006 April 19; 26(16): 4383-4393. DOI: 10.1523/JNEUROSCI.4531-05.2006.
Mohri I, Aritake K, Taniguchi H, Sato Y, Kamauchi S, Nagata N, Maruyama T, Taniike M, Urade Y. Inhibition of Prostaglandin D Synthase Suppresses Muscular Necrosis. Journal of Pathology. 2009; 174(5): 1735-1744. DOI: 10.2353/ajpath.2009.080709.
Kanaoka Y, Ago H, Inagaki E, Nanayama T, Miyano M, Kikuno R, Fujii Y, Eguchi N, Toh H, Urade Y, Hayaishi O. Cloning and Crystal Structure of Hematopoietic Prostaglandin D Synthase. Cell. 1997 September; 90(6): 1085-1095. DOI: 10.1016/S0092-8674(00)80374-8.
Okinaga T, Mohri I, Fujimura H, Imai K, Ono J, Urade Y, Taniike M. Induction of hematopoietic prostaglandin D synthase in hyalinated necrotic muscle fibers: its implication in grouped necrosis. Acta Neuropathologica. 2002; 104: 377-384.
Inoue T, Irikura D, Okazaki N, Kinugasa S, Matsumura H, Uodome N, Yamamoto M, Kumasaka T, Miyano M, Kai Y, Urade Y. Mechanism of metal activation of human hematopoietic prostaglandin D synthase. Nature Structural and Molecular Biology. 2003; 10: 291-296. DOI: 10.1038/nsb907.
High-Quality Crystallization Facility - GCF
An electron density map (below) of an alpha-amylase crystal (above) grown in space aboard ISS during Expedition 6 using the Granada Crystallization Facility.
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An electron density map (below) of an alpha-amylase crystal (above) grown on the ground during Expedition 6 using the Granada Crystallization Facility.
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Triosephosphate isomerase crystal grown in space (inset image is a close-up of the crystal) on ISS during Expedition 6 using the Granada Crystallization Facility.
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NASA Image: ISS014E20129 - A close-up view of the JAXA-Granada Crystallization Facilities (GCF) in the Commercial Generic Bioprocessing Apparatus (CGBA) during Expedition 14.
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NASA Image: ISS015E20742 - View of the Japan Aerospace Exploration Agency-Granada Crystallization Facility (JAXA-GCF) experiment hardware (GCF-B-007) in the Service Module (SM)/Zvezda. This image was taken during Expedition 15.
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NASA Image: ISS017E005001 - View of GCF-JAXA Container and GCF-JAXA Vacuum Insulator. Photo was taken during Expedition 17.
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