Protein Crystal Growth-Single Locker Thermal Enclosure System-Regulation of Gene Expression (PCG-STES-RGE) - 05.13.15
Protein crystals were grown in a temperature controlled environment. This investigation grew high quality crystals for ground-based research, which examined two proteins, one used in the food industry and the other which is used in gene expression. Science Results for Everyone
This crystal growth experiment was the longest-running to date on the International Space Station, almost three years. It focused on the crystallization of two different proteins: nucleosome core particle, a building block of chromatin responsible for gene expression and housing DNA, and D-xylose ketol-isomerase (xylose isomerase), an enzyme used by the food industry to convert glucose to fructose. Researchers examined the structure-function relationship in chromatin, and final results are pending additional sample analysis. Experiment Details
Gerald Bunick, Ph.D., Oak Ridge National Laboratory, Oak Ridge, TN, 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
November 2002 - October 2005
Previous ISS Missions
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-RGE grew two types of crystals, the nulceosome core particle and glucose isomerase.
PCG-STES-RGE (Protein Crystal Growth - Single Locker Thermal Enclosure System - Regulation of Gene Expression) was one of the nine experiments that was part of the PCG-STES suite of investigations. PCG-STES-RGE was performed in the U.S. Lab of the International Space Station.
This Expedition 6 investigation focused on the crystallization of two different proteins, nucleosome core particle and D-xylose ketol-isomerase (glucose isomerase). The nucleosome core particle is building block of chromatin, which is found in the nucleus and is responsible for gene expression and housing DNA. This investigation examined the structure-function relationship in chromatin. The glucose isomerase is an enzyme that is used in the food industry to convert glucose to fructose. The crystals produced will create a model system for the Spallation Neutron Source, an accelerator-based neutron source used for neutron-scattering research, at Oak Ridge National Laboratory.
Samples were housed in the Protein Crystal Growth - Single Locker Thermal Enclosure System using the Diffusion-Controlled Crystallization Apparatus for Microgravity (DCAM). A total of 81 DCAMs, which are slightly smaller than a 35mm film canister, were used, each contained a protein sample. Each DCAM 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 semi-permeable 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. The DCAMs were housed inside the Single-locker Thermal Enclosure System (STES).
The Single-locker Thermal Enclosure System (STES) provides a controlled-temperature environment between 1 degrees C and 40 degrees C which grew large, high-quality crystals. Its thermal control system (TCS) regulated the temperature inside the payload chamber. A fan pulled cabin air through an intake on the front panel which caused 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.
Crewmembers were required to transfer the PCG-STES from the Space Shuttle to ISS in EXPRESS Rack 4. The STES unit contained 3 trays with 27 reservoirs to house 81 DCAM cylinders. PCG-STES-RGE used two different protein samples that were part of the 81 samples.
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 DCAM chambers with the experiment samples) from the Space Shuttle Middeck to the ISS EXPRESS Rack 4. The experiment was activated on Earth where the sample and precipitant 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. The PCG-STES hardware was returned to Earth onboard STS-114.
PCG-STES is a suite of nine experiments with additional shared samples for associated investigators. Samples were taken to and from station five times for crystallization during Expeditions 2, 4, 5, and 6. The logistical considerations of space flight affected some of the results, as flight delays compromised some samples, and a jarring drop of the hardware shortly after return on 11A/STS-113 probably destroyed any larger crystals that had formed during that set of runs. PCG-STES samples in DCAM were on orbit prior to the space shuttle Columbia accident, and then spent an unprecedented 981 days (Nov 2002 - Aug 2005) on ISS before being returned on the next space shuttle flight.
The PCG-STES-RGE operated on the International Space Station during Expeditions 6 through 11. This mission was launch in November 2002 and was returned in August 2005. This was the longest running crystal experiment to date onboard the International Space Station. Further results are pending sample analysis by the investigator.
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.
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.
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.
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 Fact Sheet
Diffusion-Controlled Crystallization Apparatus for Microgravity (DCAMs). Image courtesy of NASA, Marshall Flight Space Center.
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Crystallized structure of a nucleosome core particle that was grown during a previous DCAM mission on Mir. 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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