Fact Sheet

Text Size

Protein Crystal Growth-Enhanced Gaseous Nitrogen Dewar (PCG-EGN)
12.05.12

OpNom:

Overview | Description | Applications | Operations | Results | Publications | Imagery

Experiment Overview

This content was provided by Alexander McPherson, Ph.D., and is maintained in a database by the ISS Program Science Office.

Brief Summary

Tested proteins and protein solutions that could tolerate the freeze-thaw mechanism used to initiate protein crystal experiments. Understanding these results lead to a better selection process for later protein crystal experiments on ISS.

Principal Investigator(s)

  • Alexander McPherson, Ph.D., University of California, Irvine, Irvine, CA, United States
  • Co-Investigator(s)/Collaborator(s)

  • Millard F. Reschke, Ph.D., Johnson Space Center, Houston, TX, United States
  • Anna Holmes, University of Alabama, Huntsville, Huntsville, AL, United States
  • Blake Moore, University of Alabama, Huntsville, Huntsville, AL, United States
  • Craig E. Kundrot, Ph.D., Marshall Space Flight Center, Huntsville, AL, United States
  • Dean Myles, Ph.D., European Molecular Biology Laboratory, Grenoble, France
  • Ed Meehan, Ph.D., University of Alabama, Huntsville, Huntsville, AL, United States
  • Eddie H. Snell, Ph.D., Marshall Space Flight Center, Huntsville, AL, United States
  • Ewa Ciszak, Ph.D., University of Alabama, Huntsville, Huntsville, AL, United States
  • Joseph Ng, Ph.D., University of Alabama, Huntsville, Huntsville, AL, United States
  • Laurel Karr, Ph.D., Marshall Space Flight Center, Huntsville, AL, United States
  • Liqing Chem, Ph.D., University of Alabama, Huntsville, Huntsville, AL, United States
  • Marc Pusey, Ph.D., Marshall Space Flight Center, Huntsville, AL, United States
  • Mike McFerrin, University of Alabama, Huntsville, Huntsville, AL, United States
  • Narayan Ramachandran, Ph.D., Marshall Space Flight Center, Huntsville, AL, United States
  • Robert Naumann, Ph.D., University of Alabama, Huntsville, Huntsville, AL, United States
  • Russell Judge, Ph.D., Marshall Space Flight Center, Huntsville, AL, United States
  • Xiaoyin Zhou, Ph.D., University of Alabama, Huntsville, Huntsville, AL, United States
  • Developer(s)

    University of California at Irvine, Irvine, CA, United States
    Marshall Space Flight Center, Huntsville, AL, United States

    Sponsoring Space Agency

    National Aeronautics and Space Administration (NASA)

    Sponsoring Organization

    Human Exploration and Operations Mission Directorate (HEOMD)

    ISS Expedition Duration:

    September 2000 - June 2002



    Expeditions Assigned

    0,1,2,4

    Previous ISS Missions

    PCG-EGN has flown on the following Shuttle missions to Mir : STS-71, STS-74, STS-76, STS-79, STS-81, STS-84, and STS-89.

    ^ back to top



    Experiment Description

    Research Overview

    • The primary objective of this investigation was to provide a low-cost, simple experiment platform for production of a large number of protein crystals in a microgravity environment.


    • Once returned to Earth, the crystals were analyzed for internal quality, three-dimensional structure, the factors that influence protein crystal growth nucleation, growth, order, stability, and to determine the effects of gravity on these phenomena.


    • PCG-EGN also provided fundamental data for selecting methodologies and macromolecules to be used in future liquid-liquid diffusion crystal investigations.

    Description

    Crystallization and X-ray diffraction of proteins are used to determine their structures. Understanding the structure of proteins can lead to improvements in medical treatments and pharmaceuticals for numerous conditions. The microgravity environment on board the International Space Station (ISS) is relatively free from sedimentation and convection and can provide an exceptional environment for crystal growth. Uniform, large crystals are key for determining the structure of proteins and other large biological molecules.

    The primary purpose of this experiment was to provide a simple trial platform for the production of a large number of crystals of various biological macromolecules. Thirty-two different samples of proteins were used for this experiment during four increments, the proteins are as follows:

    • Bence Jones Protein

    • Increment(s): 0, 1, 2
      Alexander McPherson, Ph.D., University of California - Irvine, Irvine, CA
      This protein is produced by patients ho have myeloma (a lymphatic cancer)

    • Canavalin mutants A293G, G924A, R301E

    • Increment(s): 0, 1, 4
      Alexander McPherson, Ph.D., University of California - Irvine, Irvine, CA
      Joseph Ng, Ph.D., University of Alabama - Huntsville, Huntsville, AL
      Canavalin is a seed storage protein found in legumes.

    • Carnitine acetyl transferase

    • Increment(s): 0
      Joseph Ng, Ph.D., University of Alabama - Huntsville, Huntsville, AL
      An enzyme that transfers short chain fatty acids from Carnitine molecules to Coenzyme A molecules.

    • Catalase

    • Increment(s): 0, 1, 2 , 4
      Alexander McPherson, Ph.D., University of California - Irvine, Irvine, CA
      A detoxifying enzyme whose function is to catalyze the breakdown of peroxide into oxygen and water in animals.

    • Concanavalin B

    • Increment(s): 0, 1
      Alexander McPherson, Ph.D., University of California - Irvine, Irvine, CA
      A plant seed protein derived from the Jack Bean. It is a defensive enzyme used to discourage attack from insects with chitin exoskeletons.

    • Cyt R Repressor

    • Increment(s): 0, 1
      Alexander McPherson, Ph.D., University of California - Irvine, Irvine, CA

    • 4?-hydroxy-tetrahydropterin dehydratase/DcoH (rat liver)

    • Increment(s): 0, 1
      Joseph Ng, Ph.D., University of Alabama - Huntsville, Huntsville, AL
      Protein that performs two functions. It performs as an enzyme that catalyzes the removal of oxygen and hydrogen through dehydration. It also performs as a transcriptional coactivator.

    • Glucose Isomerase

    • Increment(s): 0, 1
      Alexander McPherson, Ph.D., University of California - Irvine, Irvine, CA
      Russell Judge, Marshall Space Flight Center, Huntsville, AL
      A bacterial enzyme produced during the fermentation process. It is used in brewing, baking and food processing industry to convert saccharides.

    • Glutathione S-Transferase (Anopheles gambiae mosquito)

    • Increment(s): 0, 1
      Joseph Ng, Ph.D., University of Alabama - Huntsville, Huntsville, AL
      A group of enzymes capable of multiple reactions in different cellular environments.

    • Green Fluorescent Protein (Aequoria Victoria)

    • Increment(s): 0
      Marc Pusey, Ph.D., Marshall Space Flight Center, Huntsville, AL
      Protein that is found the jellyfish, Aequoria Victoria, is used to generate the green light produced by the jellyfish.

    • GTP Binding Protein

    • Increment(s): 0
      Joseph Ng, Ph.D., University of Alabama - Huntsville, Huntsville, AL
      Guanosine triphosphate binding protein (GTP) is a class of biochemical proteins and is used to relay intercellular processes.

    • Lactalbumin

    • Increment(s): 0, 1
      Alexander McPherson, Ph.D., University of California - Irvine, Irvine, CA
      Protein component of all mammalian milk.

    • Pea Lectin

    • Increment(s): 0, 2, 4
      Alexander McPherson, Ph.D., University of California - Irvine, Irvine, CA
      Joseph Ng, Ph.D., University of Alabama - Huntsville, Huntsville, AL
      Lectin is a protein that interacts with carbohydrates in order to regulate cell adhesion, glycoprotein synthesis and controls protein levels in the blood. This protein also can detect carbohydrates that are found in pathogens.

    • Lysozyme

    • Increment(s): 0, 1, 2, 4
      Alexander McPherson, Ph.D., University of California - Irvine, Irvine, CA
      A defensive enzyme that weakens bacteria cell walls, leading to their destruction.

    • Monoclonal antibody

    • Increment(s): 0, 1
      Alexander McPherson, Ph.D., University of California - Irvine, Irvine, CA
      This protein is used to treat rheumatoid arthritis.

    • Myoglobin

    • Increment(s): 0
      This protein is the oxygen-carrying molecule for muscles.

    • Osteopontin (human and rat)

    • Increment(s): 0, 1
      Joseph Ng, Ph.D., University of Alabama - Huntsville, Huntsville, AL
      Composed of carbohydrates and proteins, this protein is over expressed in a variety of cancers.

    • Phenyl preferring sulfo-transferase (human P-PST1)

    • Increment(s): 0
      Joseph Ng, Ph.D., University of Alabama - Huntsville, Huntsville, AL

    • Satellite Tobacco Mosaic Virus

    • Increment(s): 0, 2, 4
      Alexander McPherson, Ph.D., University of California - Irvine, Irvine, CA
      A virus that infects plants that causes patterns of discoloration on the leaves.

    • Thaumatin (Thaumatococcus daniellii)

    • Increment(s): 0, 1, 2, 4
      Alexander McPherson, Ph.D., University of California - Irvine, Irvine, CA
      Craig Kundrot, Ph.D., Marshal Space Flight Center, Huntsville, AL
      Joseph Ng, Ph.D., University of Alabama - Huntsville, Huntsville, AL
      Thaumatin is a sweet protein from the African serendipity berry. It is used in the food processing industry as a sugar substitute.

    • Trypsin (porcine)

    • Increment(s): 0, 1, 4
      Alexander McPherson, Ph.D., University of California - Irvine, Irvine, CA
      An enzyme that is used to break down proteins into peptides.

    • Brome Mosaic Virus

    • Increment(s): 1, 2, 4
      Alexander McPherson, Ph.D., University of California - Irvine, Irvine, CA
      BMV infects monocot plants and is produced in infected barley.

    • Goat Hemoglobin

    • Increment(s): 1
      Alexander McPherson, Ph.D., University of California - Irvine, Irvine, CA
      Hemoglobin is responsible for transporting oxygen in the blood.

    • Human Bone Alkaline Phosphatase

    • Increment(s): 1
      Laurel Karr, Ph.D., Marshall Space Flight Center, Huntsville, AL
      An enzyme crucial in bone formation and mineralization.

    • Lactate Dehydrogenase

    • Increment(s): 2
      Alexander McPherson, Ph.D., University of California - Irvine, Irvine, CA
      An enzyme responsible for converting pyruvic acid to lactic acid.

    • Pyruvate Dehydrogenase

    • Increment(s): 2
      Ewa Ciszak, Ph.D., University of Alabama-Huntsville, Huntsville, AL
      An enzyme that performs two reactions within pyruvate dehydrogenase complex: decarboxylation of pyruvate and reductive acetylation of lipoic acid.

    • Ribonuclease A

    • Increment(s): 2
      Alexander McPherson, Ph.D., University of California - Irvine, Irvine, CA
      An enzyme that cleaves (breaks) RNA into smaller pieces.

    • Gamma-E-crystallin

    • Increment(s): 4
      Dean Myles, European Molecular Biology Laboratory, Grenoble, France

    • c-phycocyanin

    • Increment(s): 4
      Naomi Chayen, Imperial College, London, UK
      The blue pigment found in blue-green algae that has fluorescent properties.

    • Cytochrome P458

    • Increment(s): 4
      Dean Myles, European Molecular Biology Laboratory, Grenoble, France
      An enzyme that transport electrons within cells.
    A secondary objective was an education program called "Student Access to Space" in which students participated in preparing some of the samples that were flown on orbit and learned about crystallization, the methods of analysis of crystals, and the impact of studies of crystals on advancing biotechnology, medicine, and basic research in structural biology. Through the Student Access to Space program, more than 500 samples were mixed by middle and high schools across the United States.

    Protein Crystal Growth-Enhanced Gaseous Nitrogen (PCG-EGN) samples were brought to ISS frozen in liquid nitrogen in a Dewar (a stainless-steel and aluminum container assembly that is similar to a Thermos bottle) at -196 degrees C (-321 degrees F) in sealed plastic capillary tubes. On board ISS, the nitrogen warmed and boiled off, turning into a gas, and the samples began to thaw. After eight days, when the samples had reached the station ambient temperature of 22 degrees C (71.6 degrees F), crystals began to form.

    ^ back to top



    Applications

    Space Applications

    Hardware that provides low-cost and low-crew maintenance crystal production in the microgravity environment is extremely beneficial to scientific studies on Earth. The crystals that are grown in microgravity grow larger and are better organized than those grown on Earth. The research that is done on these crystals may further human space exploration efforts by technological and biological advancements developed as a direct result of this research.

    Earth Applications

    Knowledge of precise three-dimensional molecular structure is a key component in biotechnology fields such as protein engineering and pharmacology. In order to obtain accurate data on the three-dimensional structure of protein crystals or other macromolecules, scientists employ a process called X-Ray Crystallography. Crystallographers construct computer models that reveal the complex structures of a protein molecule. However, in order to generate accurate computer models crystallographers must first crystallize the protein and analyze the resulting crystals by a process called X-ray diffraction. Precise measurements of thousands of diffracted intensities from each crystal help scientists map the probable positions of the atoms within each protein molecule. This complex process requires several months to several years to complete.

    On Earth, the 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. However, the microgravity environment aboard the ISS is relatively free from the effects of sedimentation and convection and provides an exceptional environment for crystal growth.

    ^ back to top



    Operations

    Operational Requirements

    PCG-EGN crew requirements were minimal, with the exception of payload transfer from the Shuttle to Station stowage space, PCG-EGN was completely self-activating.

    Operational Protocols

    The Dewar flask, filled with small crystallization samples, was frozen on Earth and housed in the passive gaseous nitrogen freezer. The samples were allowed to passively thaw in microgravity by the liquid nitrogen warming to ambient temperature and boiling off. Once the samples have reached the ambient temperature of 22 degrees C, they thaw and mix with their precipitants creating crystals. The samples are kept at 22 degrees C for the duration of the experiment. Postflight analysis included examination by microscopy, structure determination, and high-resolution refinement. Researchers conducted initial examination under an optical microscope without opening the capillary tubes. In order to conduct the additional examinations of the crystals, one of the sealed ends was cut off.

    ^ back to top



    Results/More Information

    The PCG-EGN experiment was a platform that provided an economical and potential high volume avenue to produce biological protein crystals in microgravity. The samples that are used in the EGN Dewar are placed into individual tubes, flash frozen and allowed to warm to the ambient temperature onboard ISS. Crystals produced in microgravity when compared to their counterparts grown on Earth are usually larger and more defined in structure making them better candidates for X-ray diffraction studies. The X-ray diffraction studies show researchers the structure of the molecules in the proteins and once the structure is understood, the active sites can be determined which may lead to improvement in medical treatment for certain conditions.

    Successful crystallization rates were as follows: Expedition 0 (prior to permanent human occupation of ISS), ten of 24 proteins and viruses; Expedition 1, four of 23 proteins and viruses; Expedition 2, six of eight proteins and both viruses; Expedition 4, three of nine proteins and zero of two viruses. Major crystals obtained included Bence-Jones protein, Bromegrass Mosaic Virus, canavalin, lysozyme, pea lectin, thaumatin, trypsin, and 4a-hydroxy-tetrahydropterin dehydratase (DcoH). Overall the rate of successful crystallizations was not as high as expected. Although many of the crystals produced were no better than those obtained in the ground laboratory, there were still some significant structural results.

    When compared to their Earth-grown counterparts, the space-grown thaumatin crystals diffracted to a higher resolution, and some crystals showed as much as 40% more intensity during the diffraction process. This resulted in a more accurate protein structure model (electron density map) being produced from the space-grown crystal data. The pea lectin crystals also diffracted to higher resolution than their Earth-grown counterparts. Data from the space-grown crystals were the best obtained, giving rise to the highest resolution structure for pea lectin. A refinement for the structural model of pea lectin is in progress. DcoH crystals grown on Expedition 1 also appeared to be of better quality than those grown on Earth.

    Student investigations across the four Expeditions were successful in crystallizing a number of proteins. Although many of the crystals did not appear to be better than previously analyzed crystals, some of the crystals from Expedition 2 were used for microscopic observation and X-ray examination.

    ^ back to top



    Results Publications

      Kundrot CE, Snell EH, Barnes CL.  Thaumatin crystallization aboard the International Space Station using liquid-liquid diffusion in the Enhanced Gaseous Nitrogen Dewar (EGN). Acta Crystallographica Section D: Biological Crystallography. 2002; 58(Pt 5): 751-760.
      Ciszak E, Hammons AS, Hong YS.  Use of Capillaries for Macromolecular Crystallization in a Cryogenic Dewar. Crystal Growth & Design. 2002; 2(3): 235-238. DOI: 10.1021/cg0155671.

    ^ back to top


    Ground Based Results Publications

    ^ back to top


    ISS Patents

    ^ back to top


    Related Publications

      Zörb C, Carter DC, Wright BS, Weisert A, Stapelmann J, Smolik G, 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.
      Kundrot CE, Achari A, Roeber CL, Barnes CL.  Characterization of the protein crystal growth apparatus for microgravity aboard the space station. Acta Crystallographica Section D: Biological Crystallography. 2002; 58: C375.
      Carter DC, Wright BS, Myles D, Declercq J, Ruble JR, Ho JX.  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.

    ^ back to top


    Related Websites
  • NASA Fact Sheet
  • NASA Protein Crystal Growth
  • ^ back to top



    Imagery

    image PCG-EGN hardware before launch to ISS during Expedition 2. Image courtesy of NASA, Marshall Space Center.
    + View Larger Image


    image Electron density map of thaumatin crystal grown on the International Space Station on Increment 2. Image courtesy of NASA, Marshall Space Center.
    + View Larger Image


    image NASA Image: STS110-368-018 - View of the PCG-EGN hardware in the FGB taken during STS-110.
    + View Larger Image


    image Crystal of Trypsin that was grown in microgravity during ISS Expedition 4. Image courtesy of NASA, Marshall Space Center.
    + View Larger Image


    image Catalase crystals grown in microgravity during ISS Expedition 4. Image courtesy of NASA, Marshall Space Center.
    + View Larger Image


    image Leann, a student from Ridgeview High School in Orange Park, FL, completes sample preparation as part of Student Access to Space activities. Image courtesy of NASA, Marshall Space Center.
    + View Larger Image


    Information provided by the investigation team to the ISS Program Scientist's Office.
    If updates are needed to the summary please contact JSC-ISS-Program-Science-Group. For other general questions regarding space station research and technology, please feel free to call our help line at 281-244-6187 or e-mail at JSC-ISS-Payloads-Helpline.