Japan Aerospace Exploration Agency Protein Crystallization Growth (JAXA PCG) - 12.05.18

Overview | Description | Applications | Operations | Results | Publications | Imagery

ISS Science for Everyone

Science Objectives for Everyone
The objective of the Japan Aerospace Exploration Agency Protein Crystallization Growth (JAXA PCG) investigation is to grow high quality protein crystals in microgravity. The crystals are returned to Earth to determine protein structures in detail; the structures are used to develop pharmaceutical drugs, and to explore the mystery of our lives. The protein samples are launched to the International Space Station (ISS) by a Soyuz or Progress Vehicle, and crystallized at 20℃ using the counter-diffusion method.
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.

The following content was provided by Izumi Yoshizaki, Ph.D., Shigeki Kamigaichi, and is maintained in a database by the ISS Program Science Office.
Information provided courtesy of the Japan Aerospace and Exploration Agency (JAXA).
Experiment Details

OpNom: JAXA PCG

Principal Investigator(s)
Izumi Yoshizaki, Ph.D., Japan Aerospace Exploration Agency, Tsukuba, Japan

Co-Investigator(s)/Collaborator(s)
Mitsugu Yamada, Japan Aerospace Exploration Agency, Tsukuba, Japan

Developer(s)
Japan Aerospace Exploration Agency, Tsukuba, Tsukuba, Japan

Sponsoring Space Agency
Japan Aerospace Exploration Agency (JAXA)

Sponsoring Organization
Japan Aerospace Exploration Agency

Research Benefits
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 - April 2019; -

Expeditions Assigned
19/20,21/22,23/24,27/28,29/30,33/34,35/36,39/40,41/42,43/44,45/46,47/48,49/50,51/52,53/54,55/56,57/58,59/60

Previous Missions
Inc20, 22/23, 24/25, 28, 30, 35, 39, 41, 44, 47, 49(TBD), 51(TBD)

^ back to top

Experiment Description

Research Overview

  • Aboard the International Space Station (ISS), the microgravity environment of space suppresses solution flow (convection), and allows protein crystals to grow in a stable environment. Moreover, impurities will not be delivered to the crystal surface because there is no convection. As a result, high quality protein crystals are produced.
  • The crystals are returned to Earth, and undergo X-ray structure analyses at synchrotron radiation facilities such as SPring-8. High-quality crystals obtained during the JAXA Protein Crystallization Growth (JAXA PCG) investigation on orbit exhibit a clear protein structure.
  • Detailed information on protein structures is used for designing new drugs for diseases, and for studying unknown enzyme reactions.

Description
JAXA has more than a decade of experience in protein crystal growth (PCG) experiments. JAXA has established the “Drug-design supporting platform” to contribute to promote national research for “Health and Longevity” and company businesses. In order to facilitate the use of this platform, JAXA designed a total package service, i.e., protein sample characterization, purification, crystallization screening, optimization, microgravity experiment, X-ray diffraction using synchrotron facility, and structure determination. Domestic academic users launch their sample at no charge on the condition that their research results will be published. It is fee-based for private companies (pharmaceutical companies, bio-ventures, and etc.).

^ back to top

Applications

Space Applications
This research contributes to understanding of how the microgravity environment of space can be used in a productive capacity. Accordingly, the JAXA PCG investigation uses space as a laboratory for obtaining high quality crystals. This shows how useful the ISS is for investigations of this type.

Earth Applications
This investigation contributes to the drug discovery process and basic biochemical understanding by obtaining high quality protein crystals.

^ back to top

Operations

Operational Requirements and Protocols

  • Load the protein samples into crystallization cells in Russia (Moscow) and Baikonur.
  • Install the crystallization cells into Crystallization Canister in Baikonur launch site.
  • Launch the two sets of Canisters on Soyuz/Progress.
  • After docking to the ISS, transfer the Canisters to the Japanese Pressurized Module (JPM) within 24 hours, except in the event of four-orbits docking. In the event of four-orbits docking, within 35 hours.
  • Install the Canisters into the cell tray of Protein Crystallization Research Facility (PCRF) and start the experiment for about 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 the investigation is complete, remove the Canisters from PCRF cell tray and pack for return. Retrieve on Soyuz.
  • Analyze the protein three-dimensional structure using space grown crystals at the synchrotron facility (Ex, SPring-8, Photon Factory) on the ground.

^ back to top

Decadal Survey Recommendations

Information Pending

^ back to top

Results/More Information

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.

^ back to top

Results Publications

    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.

    Sakamoto Y, Suzuki Y, Iizuka I, Tateoka C, Roppongi S, Fujimoto M, Inaka K, Tanaka H, Masaki M, Ohta K, Okada H, Nonaka T, Morikawa Y, Nakamura KT, Ogasawara W, Tanaka N.  S46 peptidases are the first exopeptidases to be members of clan PA. Scientific Reports. 2014 May 15; 4: 4977. DOI: 10.1038/srep04977. PMID: 24827749.

    Yokomaku K, Akiyama M, Morita Y, Kihira K, Komatsu T.  Core–shell protein cluster comprising haemoglobin and recombinant feline serum albumin as an artificial O2 carrier for cats. Journal of Materials Chemistry B. 2018 March 20; 6: 2417-2425. DOI: 10.1039/C8TB00211H.

    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.

    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.

    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.

    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.

    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.

    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.

    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.

    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.

    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.

^ back to top

Ground Based Results Publications

    Timofeev VI, Smirnova EA, 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.

^ back to top

ISS Patents

^ back to top

Related Publications

    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.

    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.

    Boyko KM, Timofeev VI, Samygina VR, Kuranova IP, Popov VO, Koval'chuk MV.  Protein crystallization under microgravity conditions. Analysis of the results of Russian experiments performed on the International Space Station in 2005−2015. Crystallography Reports. 2016 September; 61(5): 718-729. DOI: 10.1134/S1063774516050059.

^ back to top

Related Websites
Protein Crystal Growth on the International Space Station

^ back to top


Imagery

image Protein Crystallization Research Facility. Image courtesy of JAXA.
+ View Larger Image


image High Quality Protein Crystal Growth Experiment Canister. Image courtesy of JAXA.
+ View Larger Image


image
NASA/JAXA Image: ISS022E057676 - View of Japan Aerospace Exploration Agency (JAXA) Soichi Noguchi, Expedition 22 Flight Engineer (FE), during installation of Protein Crystal Growth (PCG) canister into the Protein Crystallization Research Facility (PCRF).

+ View Larger Image


image
NASA/JAXA Image: ISS028E049720 - Japan Aerospace Exploration Agency (JAXA) astronaut Satoshi Furukawa along with Russian cosmonaut Sergei Volkov work on the JAXA PCG investigation.

+ View Larger Image


image
NASA/JAXA Image: ISS041E111274 - View during the Japan Aerospace Exploration Agency (JAXA) Protein Crystal Growth (PCG) investigation.

+ View Larger Image


image
NASA/JAXA Image: ISS041E111301 - View of ESA astronaut Alex Gerst and Russian cosmonaut Alexander Samokutyaev (holding canisters bags) during the Japan Aerospace Exploration Agency (JAXA) Protein Crystal Growth (PCG) investigation.

+ View Larger Image


image NASA Image: ISS049E045287 - Photographic documentation taken during JAXA Protein Crystal Growth (PCG) Installation into the Protein Crystallization Research Facility (PCRF) of the Ryutai Rack.
+ View Larger Image


image
NASA Image: ISS055E004890 - Photographic documentation taken during installation of 2 JAXA PCG canisters containing over 30 protein samples into the Protein Crystallization Research Facility (PCRF) in the Ryutai Rack as part of JAXA Protein Crystal Growth (PCG) no. 14 operations.

+ View Larger Image


image
NASA Image: ISS056E075928 - View of Alex Gerst, Expedition 56 Flight Engineer (FE), taken during Protein Crystal Growth (PCG) sample retrieval from the Freezer-Refrigerator Of STirling cycle (FROST). Gerst initiates the crystallization of the samples before inserting them back into the FROST, where crystallization continues. Photo taken by Expedition 56 crew.

+ View Larger Image