Advanced Protein Crystallization Facility - Testing New Trends in Microgravity Protein Crystallization (APCF-Lysozyme) - 08.27.15
Specialized microgravity facility that offered researchers several different crystal growth options in a controlled environment that enabled undisturbed nucleation (beginning of chemical changes at discrete points in a system) and growth of proteins to obtain large crystals for analysis on Earth. Understanding the results obtained from the crystals will lead to advances in manufacturing and biological processes. Science Results for Everyone
Plants and higher animals produce lysozyme proteins as a first line of defense against harmful bacteria. Bacterial viruses also use lysozymes to facilitate infection. Since the structure of a protein and its function are directly linked, high-quality lysozyme crystals can help scientists learn more about lysozyme structure and, therefore, its role in bacterial infection. This investigation successfully produced lysozyme crystals with qualities superior to those grown on Earth. Now that the crystal structure of lysozymes has been determined, this could accelerate the design and drugs targeting specific pathogens and diseases. Experiment Details
Fermin Otalora Munoz, University of Granada, Granada, Spain
Astrium GmbH, Bremen, Germany
Sponsoring Space Agency
National Aeronautics and Space Administration (NASA)
Italian Space Agency (ASI)
ISS Expedition Duration 1
August 2001 - December 2001
Previous ISS Missions
The APCF has flown on several Shuttle flights dating back to 1985 including, STS-107 (Columbia), which was lost in 2003.
- The APCF hardware contained 8 separate protein crystal investigations; these included APCF-Camelids, APCF-Crystal Quality, APCF-Crystal Growth, APCF-Lipoprotein, APCF-Lysozyme, APCF-Octarellins, APCF-PPG10 and APCF-Rhodopsin.
- Lysozymes are proteins produced by plants and higher organisms as a first line of defense against harmful bacteria and are also used by bacterial viruses to facilitate infection. The structure of a protein and its function are directly linked. The high-quality crystals produced by APCF-Lysozyme will help scientists learn more about lysozymes' structure and thus more about its role in bacterial infection.
Understanding proteins is basic to understanding the processes of living things. While we know the chemical formulae of proteins, learning the chemical structure of these macromolecules is more difficult. Mapping the three-dimensional structure of proteins, DNA, ribonucleic acid (RNA), carbohydrates, and viruses provides information concerning their functions and behavior. This knowledge is fundamental to the emerging field of rational drug design, replacing the trial-and-error method of drug development. Microgravity provides a unique environment for growing crystals, an environment that is free of the gravitational properties that can crush the delicate structures of crystals. Currently, several test facilities are used to grow crystals.
The Advanced Protein Crystallization Facility (APCF) can support three crystal-growth methods: liquid-liquid diffusion, vapor diffusion, and dialysis. Liquid-liquid diffusion was not used during Expedition 3. In the vapor diffusion method, a crystal forms in a protein solution as a precipitant draws moisture in a surrounding reservoir. In the dialysis method, salt draws moisture away from the protein solution via a membrane separating the two, forming crystals. ESA has announced that due to potential difficulties with the vapor diffusion method that could cause experiment failure, it will no longer propose the use of this method with the APCF.
APCF-Lysozyme was one of eight protein crystal investigations that was conducted in the Advanced Protein Crystallization Facility onboard the ISS during Expedition 3. Lysozymes are produced by plants and higher organisms as a first line of defense against harmful bacteria. Lysozymes are also used by bacterial viruses to facilitate infection or to release virions (a complete virus particle that consists of an RNA or DNA core with a protein coat sometimes with external envelopes and that is the extracellular infective form of a virus) at the end of an infection cycle. The structure of a protein and its function are directly linked. The study of high-quality lysozyme crystals will help scientists learn more about its structure and thus more about its role in bacterial infection.
The crystals that are grown in microgravity are able to grow larger and better organized than ones 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 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 enough quality or large enough size. Consequently, the growth of high quality macromolecular crystals for diffraction analyses 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 medicines.
The APCF consisted of a processing chamber and an array of support systems including power and data electronics, thermal control system, and video equipment. The APCF processing chamber accommodated 48 modular reactors. Designed to fit into a single EXPRESS locker, the reactors were activated and deactivated in groups of 12 by electronic motors, allowing groups of experiments to be started at different times during APCF's stay on Station. Ten of these reactors were observed by a high-resolution video camera, which allowed investigators to study crystal growth development. The optical system was mounted on a drive to enable direct observation of a protein chamber, 10 reactors in a sequence, 5 on each side. Five of the reactors were observed with a wide field of view, five with a narrow field. In addition, a Mach-Zender interferometer in the APCF made it possible to observe five of the reactors and to measure and visualize changes in the refractive index as the crystals grew.
The Advanced Protein Crystallization Facility was computer controlled and designed to run automatically, providing undisturbed nucleation. The crew checked the status of the facility by reading the LEDs mounted on the front panel daily. APCF required continuous and auxiliary power from the Station via EXPRESS Rack. Video and computer data was also sent via the Station computer to ground operators.
The APCF reactors were filled in Europe and shipped to the Kennedy Space Center ten days before launch. The reactors were activated after transfer to EXPRESS Rack 1 on ISS. The first processing method, vapor diffusion, allowed crystals to form inside a drop of protein solution. The second processing method, dialysis, separated the protein and salt solutions with a membrane. The facility's processing chamber was maintained at 20 degrees C and temperature data was recorded throughout the mission. Camera images in black and white were digitized and stored on the facility tape recorder. Data electronics recorded and stored other information.
On return to Earth, the protein crystals produced in the APCF were examined by crystallography and computers made the mathematical calculations needed to enable three-dimensional modeling of the proteins structures. Images from the video camera and data from the interferometer enabled investigators to study crystal growth development.
Initial analysis of crystals returned from station support the findings of earlier APCF flights: comparative crystallographic analysis indicates that space-grown crystals are superior in every way to control-group crystals grown on Earth under identical conditions (except the critical space environment). Crystals grown in microgravity generally have improved morphology, larger volume, higher diffraction limit, and lower mosaicity as compared to Earth-grown crystals. The researchers reported that the electron-density maps calculated from diffraction data contained considerably more detail, allowing them to produce more accurate three-dimensional models (Vergara, 2005).
The APCF hardware performed well during ISS Expedition 3 with very few anomalies. APCF-Camelids, APCF-Crystal Quality, APCF-Crystal Growth, APCF-Lysozyme, APCF-Octarellins and APCF-PPG10 all produced excellent quality crystals that had better resolution and other optical properties than those grown on Earth. APCF-Lipoprotein successfully produced crystals but they did not achieve the expected level of resolution. APCF-Rhodopsin had slight technical problems that prevented the formation of suitable crystals.
Crystallographic analysis indicates that the space-grown crystals are superior to the control-group crystals grown on Earth. Crystals grown in microgravity generally have improved morphology, larger volume, better optical properties, higher diffraction limit and lower mosaicity when compared to Earth-grown crystals (Vergara, 2005).
Well-diffracting crystals prepared in space have two purposes: understanding the gravity-dependent phenomena (such as nucleation and growth mechanisms) and structural determination. Each new high-resolution structure may become the start of a cascade of investigations to unravel the complexity of the cellular events like growth, division, differentiation, communication, motility, death and their role in the development of multicellular organisms. This may accelerate the structure-based design and redesign of drugs targeting pathogens, diseases and degenerative cellular processes as well as of protein and nucleic acid leading to custom enzymes, ribozymes or inhibitors to treat diseases (Lober 2002).
APCF-Lysozyme produced crystals with optical qualities superior to those grown on Earth. Their crystal structure has been determined and the results published (Vergara, 2003).
Garcia-Ruiz JM, Gonzalez Ramirez LA, Gavira JA, Otalora Munoz F. Granada Crystallisation Box: a new device for protein crystallisation by counter-diffusion techniques. Acta Crystallographica Section D: Biological Crystallography. 2002 September 26; 58(10): 1638-1642. DOI: 10.1107/S0907444902014464.
Ground Based Results Publications
Patiño Lopez LD, Decanniere K, Gavira JA, Maes D, Otalora Munoz F. Protein Experiment: Scientific Data Processing Platform for On-Flight Experiment Tuning. Microgravity Science and Technology. 2012 November 1; 24(5): 327-334. DOI: 10.1007/s12217-012-9320-y.
Zegers I, Carotenuto L, Evrard C, Garcia-Ruiz JM, De Gieter P, Gonzales-Ramires L, Istasse E, Legros J, Martial J, Minetti C, Otalora Munoz F, Queeckers P, Cedric S, Van de Weerdt C, Willaert R, Wyns L, Yourassowsky C, Dubois F. Counterdiffusion protein crystallisation in microgravity and its observation with PromISS (Protein Microscope for the International Space Station). Microgravity Science and Technology. 2006; 18-3/4: 165-169.
NASA Fact Sheet
Lysozyme crystal grown on Earth. Image provided by the Bulgarian Academy of Sciences.
+ View Larger Image
Computer generated image of a lysozyme molecule.
+ View Larger Image
NASA Image: ISS003E8171 - The Advanced Protein Crystal Facility (APCF) located in EXPRESS Rack 1 onboard ISS Expedition 3. Two other experiments, MAMS and SAMS-II, are installed below it.
+ View Larger Image