Protein Crystal Growth-Single Locker Thermal Enclosure System-Science and Applications of Facility Hardware for Protein Crystal Growth (PCG-STES-SA) - 07.14.16
Protein crystals were grown in a temperature controlled environment. This investigation focused on the PCG-STES hardware and its ability to provide an environment to produce high-quality crystals. Science Results for Everyone
These investigations focused on hardware for crystal growth: Protein Crystallization Apparatus for Microgravity (PCAM) and Diffusion-Controlled Crystallization Apparatus for Microgravity (DCAM). PCAMs have nine trays with seven wells holding a drop of protein solution and precipitant, surrounded by a reservoir filled with an absorbent fluid to draw moisture away. Crystals form as the moisture is absorbed. DCAMs contain two cylindrical chambers connected by a tunnel, one holding precipitant solution and the other the sample, covered by a semipermeable membrane controlling the rate the precipitant passes through. These investigations obtained high-resolution structural data from human albumin and human antithrombin III crystals, and new structural information is anticipated. Experiment Details
Daniel C. Carter, Ph.D., New Century Pharmaceuticals, Incorporated, Huntsville, AL, United States
NASA Marshall Space Flight Center, Huntsville, AL, United States
Sponsoring Space Agency
National Aeronautics and Space Administration (NASA)
Human Exploration and Operations Mission Directorate (HEOMD)
ISS Expedition Duration
March 2001 - August 2001; December 2001 - December 2002; November 2002 - May 2003; April 2003 - October 2005
Protein crystal growth investigations have been completed on STS-63, STS-67, STS-73, STS-83, STS-85, STS-94 and STS-95. PCG-STES investigations were conducted on ISS Increments 2 and 4 -11.
- PCG-STES is comprised of nine separate investigations. They are: Improved Diffraction Quality of Crystals (IDQC), Integral Membrane Proteins (IMP), Measurements and Modeling (MM), Mitochondrial Metabolite Transport Proteins (MMTP), Material Science (MS), Ribosome for Diffraction Properties (RDP), Regulation of Gene Expression (RGE), Science and Applications (SA), and Vapor Equilibrium Kinetics Studies (VEKS).
- PCG-STES-SA studied the facility that maintained an environment conducive to crystal growth.
- The facility was able to carry many samples for investigators and increased the overall science return for each mission.
These investigations focused on the hardware that provided a suitable environment for crystal growth in microgravity. Samples were housed in the PCG-STES and within two different types of crystallization hardware: the Protein Crystallization Apparatus for Microgravity (PCAM) or the Diffusion-Controlled Crystallization Apparatus for Microgravity (DCAM).
PCAMs consist of nine trays, each containing seven vapor-equilibration wells. The nine trays are sealed inside a cylinder. Crystals are formed by the "sitting drop" method of vapor diffusion. Each sample well holds a drop of protein solution and precipitant (salts or organic solvents, which draw water away from the protein solution) mixed together. A surrounding moat holds a reservoir, filled with an absorbent fluid, that draws moisture away from the mixed solution. Crystals form as the moisture is absorbed. A rubber seal pressed into the lip of the reservoir keeps crystals from forming on Earth or from bouncing out of their wells during transport. Each cylinder holds 63 experiments for a total of 378 experiments inside the Single-locker Thermal Enclosure System (STES). PCAM was used for all samples during Expeditions 2, 4, and 5.
DCAMs, which are slightly smaller than a 35mm film canister, each contained two cylindrical chambers that are connected by a tunnel. One chamber holds the precipitant solution and the other contains the protein sample. A thin semipermeable membrane covers the protein sample that allowed the precipitant to pass through at a controlled rate. The rate of diffusion was controlled by a porous plug that separates the two chambers. This is referred to as the liquid-liquid diffusion method. Eighty-one DCAMs, which were housed inside the STES, were used for all samples during Expedition 6.
The STES provides a controlled temperature environment between 1 degrees C and 40 degrees C in which to grow large, high-quality crystals. Its thermal control system (TCS) regulates the temperature inside the payload chamber. A fan pulls cabin air through an intake on the front panel, causing the air to flow across the heat exchanger fans and then out the rear left side of the unit. Pushbuttons and a liquid crystal display (LCD) on the front panel allow the station crew to command the unit. STES can also be commanded from the ground.
These investigations also focused on key proteins of the circulatory system such as human serum albumin, human antithrombin III, and human peroxiredoxin 5 (PRDX5). In addition, S-layer protein from Bacillus sphaericus, cytochrome p450, and c-phycocyanin from Synechococcus elongatus were flown as part of associate investigations during Expedition 5.
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 as a direct result from this research.
Protein crystal growth experiments aid the generation of computer models of carbohydrates, nucleic acids and proteins, and further advance the progress of biotechnology. Understanding these results will lead to advances in manufacturing and biological processes, both in medicine and agriculture.
Operational Requirements and Protocols
Crewmembers were required to transfer the PCG-STES from the Space Shuttle to ISS U.S. Laboratory. The STES unit contained either 6 PCAM cylinders or 81 DCAM cylinders.
Each PCAM cylinder contained nine trays with seven reservoirs in each tray. The experiment was activated by rotating the shaft end of the PCAM cylinder clockwise using a socket wrench. This caused the elastomer seal to retract allowing vapor diffusion between the protein solution and the crystallization solution, starting the experiment. For the deactivation, the cylinder was rotated counter clockwise to reseal the samples.
Each DCAM held one sample plus the precipitant. The experiment was activated as soon as the chambers were filled while on Earth. This was a fully automated experiment; no crewmembers were needed for activation or deactivation.
Crewmembers transferred the experiment hardware, PCG-STES (containing the PCAM and DCAM cylinders containing experiment samples) from the Space Shuttle Middeck to the ISS U.S. Laboratory.
The PCAM samples were activated by the crewmembers by opening the door on the STES unit and rotating the cylinder clockwise using a socket wrench. The crew checked the LCD display daily and cleaned the fan inlet when necessary. The samples were deactivated by rotating the cylinder counterclockwise using a socket wrench.
The DCAM samples were activated on Earth when samples and precipitants were added to their chambers. Even though the experiment was activated prior to launch, the rate of diffusion is so slow that the crystals did not begin to grow until several weeks later.
Decadal Survey Recommendations
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Not surprisingly, given the wide array of materials and objectives, some samples did produce large crystals, while other samples produced crystals no better than those produced on Earth. Yet other samples failed to crystallize at all.
Crystals of MnSOD, produced during Expedition 4, exhibited an 80-fold volume increase when compared to the crystals produced on Earth. The crystals that were produced in orbit ranged from small, needle-like crystals to large three-dimensional crystals. These crystals were used for Synchrotron X-ray analysis, the use of a high-energy, adjustable particle beam used for crystal diffraction. Through this analysis it was determined that the diffraction resolution and quality of data for the crystals produced in microgravity were increased when compared to the diffraction resolution of the crystals grown on Earth (Vahedi-Faridi et al. 2003).
High-resolution structural data were also obtained from human albumin and human antithrombin III crystals, and publications of new structural information is anticipated. Analyses of the samples returned in August 2005 is ongoing. (Evans et al. 2009)
Vahedi-Faridi A, Porta J, Borgstahl GE. Improved three-dimensional growth of manganese superoxide dismutase crystals on the International Space Station. Acta Crystallographica Section D: Biological Crystallography. 2003; 59(Pt 2): 385-388. DOI: 10.1107/S0907444902020310. PMID: 12554961.
Ground Based Results Publications
Declercq J, Evrard C, Carter DC, Wright BS, Etienne G, Parello J. A crystal of a typical EF-hand protein grown under microgravity diffracts X-rays beyond 0.9 Å resolution. Journal of Crystal Growth. 1999; 196(2-4): 595-601. DOI: 10.1016/S0022-0248(98)00829-X.
Carter DC, Wright BS, Miller T, Chapman J, Twigg P, Keeling K, Moody K, White M, Click J, Ruble JR, Ho JX, Adcock-Downey L, Dowling T, Chang C, Ala P, Rose J, Wang BC, Declercq J, Evrard C, Rosenberg J, Wery J, Clawson D, Wardell M, Stallings W, Stevens A. PCAM: a multi-user facility-based protein crystallization apparatus for microgravity. Journal of Crystal Growth. 1999; 196: 610-622. DOI: 10.1016/S0022-0248(98)00858-6.
Fang XW, Srividya N, Golden BL, Sosnick TR, Pan T. Stepwise conversion of a mesophilic to a thermophilic ribozyme. Journal of Molecular Biology. 2003; 330(2): 177-183. DOI: 10.1016/S0022-2836(03)00582-5. PMID: 12823959.
Linger BR, Kunovska L, Kuhn RJ, Golden BL. Sindbis virus nucleocapsid assembly: RNA folding promotes capsid protein dimerization. RNA. 2004; 10(1): 128-138. DOI: 10.1261/rna.5127104. PMID: 14681591.
Kundrot CE, Roeber CL, Achari A. Characterization of the protein crystal growth apparatus for microgravity aboard the space station. Acta Crystallographica Section D: Biological Crystallography. 2002; 58: C375.
Golden BL, Kim H, Chase E. Crystal structure of a phage Twort group I ribozyme-product complex. Nature Structural and Molecular Biology. 2005 January; 12(1): 82-89. DOI: 10.1038/nsmb868. PMID: 15580277.
Ho JX, Declercq J, Myles DA, Wright BS, Ruble JR, Carter DC. Neutron structure of monoclinic lysozyme crystals produced in microgravity. Journal of Crystal Growth. 2001; 232(1-4): 317-325. DOI: 10.1016/S0022-0248(01)01077-6.
Golden BL, Kundrot CE. RNA crystallization. Journal of Structural Biology. 2003; 142(1): 98-107. PMID: 12718923.
Zörb C, Weisert A, Stapelmann J, Smolik G, Carter DC, Wright BS, Brunner-Joos KD, Wagner G. Bacteriorhodopsin crystal growth in reduced gravity--results under the conditions, given in CPCF on board of a Space Shuttle, versus the conditions, given in DCAM on board of the Space Station Mir. Microgravity Science and Technology. 2002; 13(3): 22-29. DOI: 10.1007/BF02872073. PMID: 12206160.
NASA Fact Sheet
Protein Crystallization Apparatus for Microgravity (PCAM). Image Courtesy of NASA, Marshall Flight Space Center.
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Diffusion-Controlled Crystallization Apparatus for Microgravity (DCAMs). Image Courtesy of NASA, Marshall Flight Space Center.
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NASA Image: ISS007E14210 - Close-up of the Single-locker Thermal Enclosure System in Express Rack 4 onboard ISS, during Expedition 7.
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NASA Image: ISS005E21531 - Astronaut Peggy A. Whitson, Expedition Five science officer, works the PCG-STES hardware on board the International Space Station.
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