Protein Crystal Growth-Single Locker Thermal Enclosure System-Synchrotron Based Mosaicity Measurements of Crystal Quality and Theoretical Modeling (PCG-STES-MM) - 07.14.16
Protein crystals were grown in a temperature controlled environment. This investigation's primary objective was to grow high quality, large crystals for ground-based research which were used in X-ray diffraction studies to discern the function and structure of the proteins. Science Results for Everyone
Nine crystallization experiments with shared samples went to and from the International Space Station five times. Given the wide array of materials and objectives, it’s no surprise that some samples produced large crystals, others produced similar Earth-quality crystals, and some failed to crystallize at all. Crystals of the enzyme Manganese Superoxide Dismutase (MnSOD) increased 80-fold in volume, ranging from small, needle-like crystals to large three-dimensional ones. Crystal diffraction analysis determined an increased diffraction resolution and quality of data for crystals produced in microgravity. High-resolution structural data were also obtained from human albumin and antithrombin III crystals, with analysis still underway. Experiment Details
Gloria E.O Borgstahl, Ph.D., University of Toledo (Expedition 4) and University of Nebraska Medical Center (Expedition 5), Toledo, OH, 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
December 2001 - December 2002
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).
- The objective of PCG-STES-MM was to grow high quality, large deoxyribonucleic (DNA) binding proteins and antioxidant enzyme crystals for ground-based X-ray diffraction.
During Expedition 4, this investigation focused on the crystallization of human Replication Protein A (RPA) and manganese superoxide dismutase (SOD) to be used in ground-based X-ray diffraction studies to understand the atomic structure. Human RPA is a single-stranded DNA binding protein that is used in DNA metabolism (replication, transcription, recombination, and repair). SODs are antioxidant enzymes that protect living cells against oxide radicals that are associated with cell damage.
Samples were housed in the Protein Crystal Growth - Single Locker Thermal Enclosure System using the Protein Crystallization Apparatus for Microgravity (PCAM). The PCAMs consisted of nine trays, each containing seven vapor-equilibration wells. The nine trays were sealed inside a cylinder. Crystals were formed by the "sitting drop" method of vapor diffusion. Each sample well holds a drop of protein solution and precipitant (salts or organic solvents that draw water away from the protein solution) mixed together. A surrounding moat holds a reservoir, filled with an absorbent fluid, which draws moisture away from the mixed solution. Crystals formed as the moisture was absorbed. A rubber seal pressed into the lip of the reservoir kept the crystals from forming on Earth or from bouncing out of their wells during transport. This experiment had a total of 168 samples onboard ISS during two Expeditions inside the Single-locker Thermal Enclosure System (STES), making this an ideal method for mass crystal production.
The Single-locker Thermal Enclosure System (STES) provided a controlled-temperature environment between 1 degrees C and 40 degrees C to grow large, high-quality crystals. The thermal control system (TCS) regulated the temperature inside the payload chamber. Cabin air was pulled through an intake fan located 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 an LCD display on the front panel allowed the crew to command the unit.
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.
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 data 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.
Operational Requirements and Protocols
Crewmembers were required to transfer the PCG-STES from the Space Shuttle Middeck to ISS in EXPRESS Rack 4. Expeditions 4 used 4 STES units containing six PCAM cylinders in each unit. Each cylinder contained nine trays with seven reservoirs in each tray. PCG-STES-MM had 35 samples that were launched on December 5, 2001 and returned on April 19, 2002. Expedition 5 used 1 STES containing six PCAM cylinders. PCG-STES-MM had 133 samples for two proteins that were launched on October 18, 2002 and returned on December 7, 2002.
The experiment was activated by rotating the shaft end of the PCAM cylinder clockwise using a socket wrench. This causes the elastomer seal to retract allowing vapor diffusion between the protein solution and the crystallization solution, starting the experiment. For the deactivation, the cylinder is rotated counter clockwise to reseal the samples.
Crewmembers transferred the experiment hardware, PCG-STES (containing the PCAM chambers with the experiment samples) from the Space Shuttle Middeck to the ISS EXPRESS Rack 4. The experiment was 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. For the return flight, the PCG-STES hardware was returned to the Shuttle for transport to Earth.
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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
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.
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.
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.
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.
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.
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.
Golden BL, Kundrot CE. RNA crystallization. Journal of Structural Biology. 2003; 142(1): 98-107. PMID: 12718923.
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.
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.
NASA Fact Sheet
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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Image from Vahedi-Faridi, A. Acta Crystallographica, Section D, Biological Crystallography, 2003. Shows a Manganese Superoxide dismutases (MnSOD) crystal grown in microgravity. The pink color is a result from the oxidized manganese in the active site.
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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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