Protein Crystal Growth-Single Locker Thermal Enclosure System-Engineering a Ribozyme for Diffraction Properties (PCG-STES-RDP) - 11.22.16
Protein crystals were grown in a temperature controlled environment. This investigation's primary objective was to grow high quality crystals for ground-based research which were to be used in x-ray crystallography of the active site of ribonucleic acid (RNA) enzyme. Science Results for Everyone
This investigation attempted to grow crystals from an engineered RNA enzyme. None were obtained the first run, perhaps because the hexylene glycol precipitant evaporated more than expected. Analysis also showed increased concentrations of magnesium chloride, which may have led to sample degradation. After the second run, detailed examination revealed growth of tiny crystals that may have been overlooked initially and could have grown in orbit or post-landing. It also appeared that the samples had been disturbed from dropping or jolting the unit. This work did lead to an increase in ground growth of RNA enzyme crystals from 4 to 50 percent. Experiment Details
Barbara L. Golden, Ph.D., Purdue University, West Lafayette, IN, 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).
- The objective of PCG-STES-RDP was to grow high-quality ribonucleic acid (RNA) enzyme crystals for ground-based X-ray crystallography studies.
PCG-STES-RDP (Protein Crystal Growth - Single Locker Thermal Enclosure System - Engineering a Ribozyme for Diffraction Properties) is one of the nine experiments that was part of the PCG-STES suite of investigations. PCG-STES-RDP was performed in the U.S. Lab of the International Space Station during Expedition 5. This investigation focused on the crystallization of a molecular engineered ribonucleic acid (RNA) enzyme to be used in x-ray crystallography to view the active site on the RNA enzyme.
RNA enzyme (also called a ribozyme) is an RNA molecule that is responsible for catalyzing their own cleavage or cleavage of other RNA strands. For this investigation, a ribozyme was engineered to be used to examine the Group I introns (segment of RNA that is not coded for a gene) through x-ray crystallography in order to determine structure and function. These areas are spliced out of the messenger RNA before it moves out of the cell nucleus. Group I introns are capable of removing themselves without the assistance of additional enzymes.
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 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. This experiment had a total of 70 samples onboard ISS during two experiments runs 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 temperature-controlled 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. Six PCAM cylinders were used. Each cylinder contained nine trays with seven reservoirs in each tray. PCG-STES-RDP used 35 samples on two separate runs for a total of 70 samples during Expedition 5. The first run of the experiment began in June 2002 and concluded in October 2002. The second run, with 35 fresh samples, began in October 2002 and concluded in December 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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PCG-STES-RDP operated on ISS during Expedition 5 during two separate runs. The experiment used an engineered RNA enzyme sample to grow crystals. Crystals were not obtained from the first run of this investigation. This was attributed to using the precipitant MPD (hexylene glycol), which is now known to evaporate more than expected in the PCAMs. This investigation began in June 2002 and was concluded in October 2002. During the post analysis, it was discovered the there was an increase in the concentration of magnesium chloride in the samples, which may have lead to the degradation of the samples. (One Year Postflight Report, 2003)
The second run of this investigation began in October 2002 and concluded in December 2002. The detailed examination of the PCAM trays revealed that tiny crystals had grown in the trays. They may have been overlooked in the initial examination when the samples were returned to Earth. It is not known whether the crystals grew in orbit or post-landing. Upon further examination, it appeared that the samples had been disturbed. An explanation of the disturbance is that the STES unit had been dropped or jolted during transport with enough force to displace the samples or shatter crystals.
These experiments lead to an optimization of growing the ribozyme in ground laboratories. Crystals are now more reproducible. The rate of growth has increased from 4% to 50%. (One Year Postflight Report, 2003)
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.
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.
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, Chase E. Crystallization and preliminary diffraction analysis of a group I ribozyme from bacteriophage twort. Acta Crystallographica Section F: Structural Biology and Crystallization Communications. 2005 January 1; 61(1): 71-74. DOI: 10.1107/S1744309104028337. PMID: 16508095.
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.
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.
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.
Golden BL, Kundrot CE. RNA crystallization. Journal of Structural Biology. 2003; 142(1): 98-107. PMID: 12718923.
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.
Paukstelis PJ, Chen J, Chase E, Lambowitz AM, Golden BL. Structure of a tyrosyl-tRNA synthetase splicing factor bound to a group I intron RNA. Nature. 2008 January 3; 451(7174): 94-97. DOI: 10.1038/nature06413. PMID: 18172503.
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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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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