Protein Crystal Growth-Single Locker Thermal Enclosure System-Improved Diffraction Quality of Crystals (PCG-STES-IDQC) - 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
Delays, drops, and disasters affected these crystal growth experiments. Flight delays compromised some samples, dropped hardware upon return affected another, and the space shuttle Columbia accident left another orbiting on the International Space Station for an unprecedented 981 days. In one experiment growing basic fibroblast growth factor (bFGF) and thaumatin crystals, 8 of 18 chambers produced crystals, the largest about 80 micrometers by 50 micrometers. Thaumatin samples had to be re-formulated and a recipe transcribing error affected four sample chambers, with 9 of the remaining chambers producing large, needle splay crystals. Experiment Details
Craig E. Kundrot, Ph.D., Marshall Space Flight Center, 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-IDQC is to obtain superior protein crystals for ground-based X-ray diffraction studies.
- The secondary objective was to determine if some chemical conditions that do not produce crystals on Earth would produce crystals in space.
PCG-STES-IDQC (Improved Diffraction Quality of Crystals) is one of the nine experiments that was part of the PCG-STES suite of investigations. PCG-STES-IDQC was performed in the U.S. Lab of the International Space Station.
These experiments focused on the growth of better quality crystals for X-ray diffractions analysis. During ISS Expedition 2, two types of samples were selected. The first was a complex of basic fibroblast growth factor (bFGF) (a fibroblast is a cell from which connective tissue develops) and 19t2mod, a 42-nucleotide DNA that inhibits bFGF activity (inhibition of bFGF activity is a type of anti-cancer therapy). The second sample type was the plant protein thaumatin. Thaumatin, which is a protein from the katemfe fruit of West Africa, is an extremely potent sweetener. The objective of this sample type was to determine whether some of the chemical conditions that do not produce crystals on Earth would produce crystals in microgravity. In addition to the samples above, two further proteins, rDerf2 and glucocerebrosidase, were flown as part of an associate investigation during Expeditions 4 and 5. A deficiency of beta-glucocerebrosidase leads to Gaucher's disease, which is a rare chronic disorder of cerebroside metabolism that is characterized by enlargement of the spleen, skin pigmentation, and bone lesions.
Both 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 basic fibroblast growth factor (bFGF) complex was loaded into 18 chambers. Thaumatin was loaded into 45 chambers.
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 22 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 22 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-IDQC operated on the International Space Station during Expedition 2. The experiment used two types of protein samples, basic fibroblast growth factor (bFGF) and thaumatin, used to grow crystals and was active for 22 days. The bFGF samples were originally supposed to be loaded into 45 PCAM chambers. Due to regulations that are related to flight, the original formulation of chemicals was declared ineligible for fight. After a new set a chemical formulations were created, 18 chambers were loaded with the samples. Of the 18 chambers 8 produced crystals. The largest crystal was about 80 micrometers x 50 micrometers. The thaumatin samples were originally to be loaded into 23 PCAM chambers. Due to flight regulations, one compound (hexadecyltimethyl ammonium bromide) in the sample was not eligible for flight. A new formulation was created to replace the original sample and was used to fill 45 chambers. There was a clerical error during the transcribing of the recipe and this affected 4 sample chambers. Of the remaining chambers, 9 produced crystals. The crystals that were produced in microgravity were large needle splays (Kundrot, Increment Two One Year Postflight Report).
Ground Based Results Publications
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.
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.
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.
NASA Fact Sheet
Image shows crystals that were produced in microgravity from a complex of basic fibroblast growth factor (bFGF). Image courtesy of NASA, Marshall Space Center.
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Image shows crystals from the plant protein thaumatin that were grown in ground-based experiments. Image courtesy of NASA, Marshall Space Center.
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Image shows crystals from the plant protein thaumatin that were grown onboard ISS. Image courtesy of NASA, Marshall Space Center.
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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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