Protein Crystal Growth-Single Locker Thermal Enclosure System-Crystallization of the Mitochondrial Metabolite Transport Proteins (PCG-STES-MMTP) - 07.14.16
Protein crystals were grown in a temperature controlled environment. This investigation grew high quality crystals for ground-based research, which examined the proteins that are used in transporting carbon into cells. Science Results for Everyone
Nine experiments attempted to grow crystals in microgravity over several expeditions. Due to the Columbia accident, samples from the Protein Crystal Growth-Single Locker Thermal Enclosure System-Regulation of Gene Expression (PCG-STES) experiment were on-board the International Space Station an unprecedented 981 days before retrieval. The objective of PCG-STES-MMTP was to grow crystals of mitochondrial metabolite proteins (MMTP). The investigation suffered loss of some of the samples during the mission. Crystals did grow in four out of 28 wells of the Diffusion-Controlled Crystallization Apparatus for Microgravity, but these were too small and of insufficient quality to perform x-ray diffraction. Experiment Details
Ronald Kaplan, Ph.D., The Chicago Medical School, Chicago, IL, 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
June 2002 - 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).
- PCG-STES-MMTP grew superior quality protein crystals for ground-based X-ray diffraction studies of mitochondrial metabolite proteins.
PCG-STES-MMTP (Protein Crystal Growth - Single Locker Thermal Enclosure System - Crystallization of the Mitochondrial Metabolite Transport Proteins) is one of the nine experiments that was part of the PCG-STES suite of investigations. PCG-STES-MMTP was performed in the U.S. Lab of the International Space Station.
This Expedition 5 investigation focused on the crystallization of the mitochondrial citrate transporter protein (CTP). Mitochondria are round or rod-shaped organelles that are located in most cells and produce enzymes for the metabolic conversion of food to energy (citric acid cycle). CTP is located in the mitochondrial inner membrane and it is responsible for transporting citrate across this membrane. In humans and other higher animals, CTP is an important part in cellular metabolism by transporting carbon into the cells, where it fuels fatty acid and the cholesterol synthesis pathways.
Samples were housed in the Protein Crystal Growth - Single Locker Thermal Enclosure System using the Protein Crystallization Apparatus for Microgravity (PCAM). 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 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 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), making this an ideal system for producing sufficient numbers of crystals for analysis.
The Single-locker Thermal Enclosure System (STES) provides a controlled-temperature environment between 1 degrees C and 40 degrees C to grow large, high-quality crystals. It's 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 an LCD display on the front panel allow the crew to command the unit. STES can also be commanded from the ground.
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 are required to transfer the PCG-STES from the Space Shuttle to ISS in EXPRESS Rack 1. Six PCAM cylinders were used. Each cylinder contained nine trays with seven reservoirs in each tray. PCG-STES-MMTP had 35 samples.
The experiment is 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 1. 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.
Decadal Survey Recommendations
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The PCG-STES-MMTP operated on the International Space Station during Expedition 5. The experiment used Mitochondrial Metabolite Transport Proteins samples to grow crystals. This investigation suffered sample loss from the sample pedestals during the mission. Even though there was a loss of some of the sample, crystals did grow in four out of the twenty-eight wells. Unfortunately, these crystals were too small and poor quality to perform x-ray diffraction.
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
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.
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.
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.
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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