Stanley Koszelak

Protein and virus crystal growth on International Microgravity Laboratory-2

Two T = 1 and one T = 3 plant viruses, along with a protein, were crystallized in microgravity during the International Microgravity Laboratory-2 (IML-2) mission in July of 1994. The method used was liquid-liquid diffusion in the European Space Agencys Advanced Protein Crystallization Facility (APCF). Distinctive alterations in the habits of Turnip Yellow Mosaic Virus (TYMV) crystals and hexagonal canavalin crystals were observed. Crystals of cubic Satellite Tobacco Mosaic Virus (STMV) more than 30 times the volume of crystals grown in the laboratory were produced in microgravity. X-ray diffraction analysis demonstrated that both crystal forms of canavalin and the cubic STMV crystals diffracted to significantly higher resolution and had superior diffraction properties as judged by relative Wilson plots. It is postulated that the establishment of quasi-stable depletion zones around crystals growing in microgravity are responsible for self-regulated and more ordered growth.

Incorporation of impurities into macromolecular crystals

The chemical, mechanical and diffraction properties of crystals grown from solution, as well as their growth kinetics and morphological development, depend very much on the types and concentrations of impurities present in their mother liquor. The situation appears vastly more complicated in the case of macromolecular crystals because of the complex nature of the molecules and the biochemical milieu from which they are derived. An attempt is made here to catalog and characterize these various impurities. One class of impurities, large foreign particles (such as dust), microcrystals, misoriented three-dimensional nuclei, and large molecular clusters has been investigated in detail using atomic force microscopy. With this technique we have directly visualized the incorporation of such larger impurities and have delineated some of their more striking consequences. In particular we have found that in some cases such incorporation is accompanied by visible defect formation or dislocations. In other cases of small three-dimensional nuclei, coalescence proceeds in a smooth manner, with alignment and knitting together of the respective lattices. A calculation of the overall defect density in canavalin crystals shows the number of defects to be many orders of magnitude greater than found for most conventional crystals.

Protein crystal growth results for shuttle flights STS-26 and STS-29

Abstract Recent advances in protein crystallography have significantly shortened the time and labor required to determine the three-dimensional structures of macromolecules once good crystals are available. Crystal growth has become a major bottleneck in further development of protein crystallography. Proteins and other biological macromolecules are notoriously difficult to crystallize. Even when usable crystals are obtained, the crystals of essentially all proteins and other biological macromolecules are poorly ordered, and diffract to resolutions considerably lower than that available for most crystals of simple organic and inorganic compounds. One promising area of research which is receiving widespread attention is protein crystal growth in the microgravity environment of space. A series of protein crystal growth experiments were performed on US shuttle flight STS-26 in September 1988 and STS-29 in March 1989. These proteins had been studied extensively in crystal growth experiments on earth prior to the microgravity experiments. For those proteins which produced crystals of adequate size, three-dimensional intensity data sets with electronic area detector systems were collected. Comparisons of the microgravity-grown crystals with the best earth-grown crystals obtained in numerous experiments demostrate that the microgravity-grown crystals of these proteins are larger, display more uniform morphologies, and yield diffraction data to significantly higher resolutions. Analyses of the three-dimensional data sets by relative-Wilson plots indicate that the space-grown crystals are more highly ordered at the molecular level than their earth-grown counterparts.

Crystallization of biological macromolecules from flash frozen samples on the Russian Space Station Mir.

One hundred eighty‐three flash frozen, liquid–liquid diffusion and batch method protein and virus crystallization samples were launched aboard the Space Shuttle Discovery on June 27 (STS‐71) and transferred to the Russian Space Station Mir on July 1, 1995. They were returned to earth November 20, 1995 (STS‐74). Subsequent examination showed that of the 19 types of proteins and viruses investigated, 17 were crystallized during the period on Mir. The experiment demonstrates the utility of this very simple and inexpensive approach for the crystallization of biological macromolecules in space over extended time periods. The distribution of crystals among the three types of containers used indicated small samples yielded results equal or better than larger samples and that long diffusion path lengths were clearly better. Distribution of crystals within the container tubes showed a striking gradient of quality and size that indicated long, narrow tubes yield superior crystals, as predicted from other work based on crystallization in capillaries.

Preliminary analysis of crystals of satellite tobacco mosaic virus.

Satellite tobacco mosaic virus (STMV), a small T = 1 icosahedral plant virus, has been crystallized in a form suitable for high-resolution X-ray diffraction analysis. The crystals, which diffract to better than 2.5 A resolution, are of space group I222 and have unit cell dimensions of a = 176 A, b = 192 A and c = 205 A. The centers of the virus particles occupy 222 symmetry points in the unit cell and one quarter of the virus particle constitutes the asymmetric unit, which is therefore comprised of 15 capsid protein molecules. From packing considerations, the maximum diameter of the STMV particles cannot exceed 165 A, and it is probably 5 to 10 A less than this value.

Protein crystal growth rates determined by time lapse microphotography

Abstract Time lapse video microscopy has been used to make qualitative observations of the events that transpire during normal and abnormal protein crystal growth. It has also been used to make quantitative assessments of growth rates for a variety of different protein crystals. From analyses of the growth rates, we have estimated that in the most rapidly growing crystals we have recorded, as many as 20 layers of protein molecules add to a single crystal face per second. In the slowest cases of growth, such as virus crystals, a minute or more may be required for addition of a single layer. In almost all cases, growth was linear over nearly the entire period of growth before leveling near growth termination. We present here a small but typical sample of the results obtained using the time lapse video microscopy technique.

The effects of neutral detergents on the crystallization of soluble proteins

Abstract A number of protein and nucleic acid molecules were crystallized in the presence and the absence of the neutral detergent β-octyl glucoside (0.0–1.5%) from polyethylene glycol and ammonium sulfate. Our results, though in great part qualitative, support the contention that the growth of macromolecular crystals is influenced in a positive manner by the presence of detergent. In general, more reproducible and rapid growth was observed with a larger proportion of large individual crystals at the expense of microcrystals. In some instance, the crystal habit or the crystallographic unit cell was altered.

Time lapse microphotography of protein crystal growth using a color VCR

Abstract A time lapse video color recorder (VCR) interfaced to a color video camera mounted on a light microscope was used to observe and record the growth of three protein crystals: lysozyme, canavalin, and a fungal protease. The studies produced a number of interesting observations regarding the formation of crystal habit, crystal aggregates, and crystal polymorphs. It further provided a simple and easy means for the measurement of growth rates. The system is described here along with a brief collection of some of our conclusions based on the time lapse video sequences.

Microgravity protein crystal growth; results and hardware development

Abstract Protein crystal growth experiments have been performed on a series of US shuttle missions. Crystallographic studies of proteins and nucleic acids have played key roles in establishing the structural foundations of molecular biology and biochemistry and for revealing structure/function relationships that are of major importance in understanding how macromolecules operate in biological systems. A number of major advances in the technology involved in determining protein structures have shortened the time span involved in structure determination. The major bottleneck in the widespread application of protein crystallography is the ability to produce high quality crystals that are suitable for a complete structural analysis. Evidence from several investigations indicates that crystals of superior quality can be obtained in a microgravity environment. This paper summarizes results obtained from a series of US shuttle missions and describes new hardware currently being developed for future shuttle missions.

Crystallization and preliminary X-ray analysis of the vitamin D-binding protein from human serum.

The vitamin D-binding protein from human serum has been crystallized from polyethylene glycol as a complex with 25-hydroxyvitamin D3 and examined by X-ray diffraction photography. The space group of the crystals is C2 with a = 203.0 A, b = 75.8 A, c = 90.9 A and beta = 109.5 degrees. There are two molecules of 56,000 dalton in the asymmetric unit. The crystals diffract to about 3.0 A resolution but the patterns exhibit a substantial level of diffuse scatter.