Protein Crystal Growth-Single Locker Thermal Enclosure System-Vapor Equilibrium Kinetics Studies (PCG-STES-VEKS) - 07.14.16
Protein crystals are grown in a temperature controlled environment. This investigation will obtain high quality crystal for ground-based research. Study of protein crystals is essential for visualizing proteins and developing new drugs and agricultural products. Science Results for Everyone
To figure out how long crystallization experiments take to reach equilibrium, this one used ammonium sulfate, sodium chloride, 2-methyl-2,4-pentanediol, and polyethylene glycol. The ammonium sulfate and sodium chloride crystals showed equilibrium in four days and 90 percent equilibrium in two days, indicating that 10-day missions will be sufficient. For the remaining samples, time to equilibrium was dependent on drop volume, with the highest volume taking approximately 10 days. On Expedition 5, samples in seven of nine trays lost volume during the experiment, likely due to an unexpected extension of the mission. Due to the volume loss, kinetics data were not obtained. Experiment Details
Aniruddha Achari, Ph.D., Raytheon, 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
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 primary objective of PCG-STES-VEKS is to characterize the time crystallization experiments take to reach equilibrium.
- The results were to help future investigators choose precipitants that would optimize crystallization conditions for their experiments.
PCG-STES-VEKS (Vapor Equilibrium Kinetics Studies) is one of the nine experiments that was part of the PCG-STES suite of investigations. PCG-STES-VEKS was performed in the U.S. Lab of the International Space Station.
These experiments focused on the vapor equilibrium kinetic studies designed to characterize the time crystallization experiments take to reach equilibrium in microgravity. This investigation had 378 samples that used Ammonium Sulfate, Sodium Chloride, 2-Methyl-2,4-Pentanediol and Polyethylene Glycol as precipitants activated through the sitting drop method. The experiments gathered data for a series of days after activation. The samples had different droplet volumes and precipitants. The results of this investigation were intended for future experimenters using the Protein Crystallization Apparatus for Microgravity (PCAM) hardware to help them choose the precipitants that could optimize crystallization conditions for growing large molecules.
The samples were loaded into six PCAMS where there were activated for 39, 18, 10, 4 and 2 days. One PCAM was never activated on orbit.
The 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 method for mass crystal production.
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. 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 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. 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. This experiment was active for 39, 18, 10, 4 and 2 days on ISS.
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 experiment operated autonomously for approximately 39, 18, 10, 4 and 2 days. 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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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-VEKS operated on the International Space Station during Expeditions 2, 4, 5. The experiment used precipitants of ammonium sulfate, sodium chloride, 2-methyl-2,4-pentanediol and polyethylene glycol. The samples that used ammonium sulfate and sodium chloride showed that activation of four days provides equilibrium, while activation for two days provides 90% equilibrium. This indicates that Shuttle missions of 10 days are long enough to allow salt samples to reach equilibrium. For the remaining samples, time to equilibrium is dependent on the drop volume; at the highest volume, it will take approximately 10 days to reach equilibrium. (Achari, Increments 2 and 4 One Year Postflight Reports).
Expedition 5 used nine trays in PCAM 6 on flight STS-111/UF2. The sample in seven of the nine trays lost volume during the experiment. The Space Shuttle mission was unexpectedly extended which might explain the loss of volume in the trays. Due to the loss of volume, the kinetics data was not obtained. (Achari, Increment 5 One Year Postflight Report).
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.
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