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NASA NTRS2003

Growing Larger Crystals for Neutron Diffraction

Obtaining crystals of suitable size and high quality has been a major bottleneck in macromolecular crystallography. With the advent of advanced X-ray sources and methods the question of size has rapidly dwindled, almost to the point where if one can see the crystal then it was big enough. Quality is another issue, and major national and commercial efforts were established to take advantage of the microgravity environment in an effort to obtain higher quality crystals. Studies of the macromolecule crystallization process were carried out in many labs in an effort to understand what affected the resultant crystal quality on Earth, and how microgravity improved the process. While technological improvements are resulting in a diminishing of the minimum crystal size required, neutron diffraction structural studies still require considerably larger crystals, by several orders of magnitude, than X-ray studies. From a crystal growth physics perspective there is no reason why these 'large' crystals cannot be obtained: the question is generally more one of supply than limitations mechanism. This talk will discuss our laboratory s current model for macromolecule crystal growth, with highlights pertaining to the growth of crystals suitable for neutron diffraction studies.

Pusey, Marc↗
NASA NTRS2004

Macromolecular Crystallization in Microgravity

The key concepts that attracted crystal growers, macromolecular or solid state, to microgravity research is that density difference fluid flows and sedimentation of the growing crystals are greatly reduced. Thus, defects and flaws in the crystals can be reduced, even eliminated, and crystal volume can be increased. Macromolecular crystallography differs from the field of crystalline semiconductors. For the latter, crystals are harnessed for their electrical behaviors. A crystal of a biological macromolecule is used instead for diffraction experiments (X-ray or neutron) to determine the three-dimensional structure of the macromolecule. The better the internal order of the crystal of a biological macromolecule then the more molecular structure detail that can be extracted. This structural information that enables an understanding of how the molecule functions. This knowledge is changing the biological and chemical sciences with major potential in understanding disease pathologies. Macromolecular structural crystallography in general is a remarkable field where physics, biology, chemistry, and mathematics meet to enable insight to the basic fundamentals of life. In this review, we examine the use of microgravity as an environment to grow macromolecular crystals. We describe the crystallization procedures used on the ground, how the resulting crystals are studied and the knowledge obtained from those crystals. We address the features desired in an ordered crystal and the techniques used to evaluate those features in detail. We then introduce the microgravity environment, the techniques to access that environment, and the theory and evidence behind the use of microgravity for crystallization experiments. We describe how ground-based laboratory techniques have been adapted to microgravity flights and look at some of the methods used to analyze the resulting data. Several case studies illustrate the physical crystal quality improvements and the macromolecular structural advances. Finally, limitations and alternatives to microgravity and future directions for this research are covered.

Snell, Edward H.↗
NASA NTRS2000

Fluid Physics and Macromolecular Crystal Growth in Microgravity

The first protein crystallization experiment in microgravity was launched in April, 1981 and used Germany's Technologische Experimente unter Schwerelosigkeit (TEXUS 3) sounding rocket. The protein P-galactosidase (molecular weight 465Kda) was chosen as the sample with a liquid-liquid diffusion growth method. A sliding device brought the protein, buffer and salt solution into contact when microgravity was reached. The sounding rocket gave six minutes of microgravity time with a cine camera and schlieren optics used to monitor the experiment, a single growth cell. In microgravity a strictly laminar diffusion process was observed in contrast to the turbulent convection seen on the ground. Several single crystals, approx 100micron in length, were formed in the flight which were of inferior but of comparable visual quality to those grown on the ground over several days. A second experiment using the same protocol but with solutions cooled to -8C (kept liquid with glycerol antifreeze) again showed laminar diffusion. The science of macromolecular structural crystallography involves crystallization of the macromolecule followed by use of the crystal for X-ray diffraction experiments to determine the three dimensional structure of the macromolecule. Neutron protein crystallography is employed for elucidation of H/D exchange and for improved definition of the bound solvent (D20). The structural information enables an understanding of how the molecule functions with important potential for rational drug design, improved efficiency of industrial enzymes and agricultural chemical development. The removal of turbulent convection and sedimentation in microgravity, and the assumption that higher quality crystals will be produced, has given rise to the growing number of crystallization experiments now flown. Many experiments can be flown in a small volume with simple, largely automated, equipment - an ideal combination for a microgravity experiment. The term "protein crystal growth" is often historically used to describe these microgravity experiments. This is somewhat inaccurate as the field involves the study of many varied biological molecules including viruses, proteins, DNA, RNA and complexes of those structures. For this reason we use the term macromolecular crystal growth. In this chapter we review a series of diagnostic microgravity crystal growth experiments carried out principally using the European Space Agency (ESA) Advanced Protein Crystallization Facility (APCF). We also review related research, both experimental and theoretical, on the aspects of microgravity fluid physics that affect microgravity protein crystal growth. Our experiments have revealed some surprises that were not initially expected. We discuss them here in the context of practical lessons learnt and how to maximize the limited microgravity opportunities available.

Helliwell, John R.↗
NASA NTRS2003

Hot Views on Cold Crystals: The Application of Thermal Imaging in Cryo-crystallography

In the past we have used thermal imaging techniques to visualize the cryocooling processes of macromolecular crystals. From these images it was clear that a cold wave progresses through a crystal starting at the face closest to the origin of the cold stream and ending at the point furthest away. During these studies we used large volume crystals, which were clearly distinguished from the loop holding them. These large crystals, originally grown for neutron diffraction studies, were chosen deliberately to enhance the imaging. As an extension to this work, we present used thermal imaging to study small crystals, held in a cryo-loop, in the presence of vitrified mother liquor. The different infrared transmission and reflectance properties of the crystal in comparison to the mother liquor surrounding it are thought to be the parameter that produces the contrast that makes the crystal visible. An application of this technology may be the determination of the exact location of small crystals in a cryo-loop. Data from initial tests in support of application development was recorded for lysozyme crystals and for bFGF/dna complex crystals, which were cryo-cooled and imaged in large loops, both with visible light and with infrared radiation. The crystals were clearly distinguished from the vitrified solution in the infrared spectrum, while in the case of the bFGF/dna complex the illumination had to be carefully manipulated to make the crystal visible in the visible spectrum. These results suggest that the thermal imaging may be more sensitive than visual imaging for automated location of small crystals. However, further work on small crystals robotically mounted at SSRL did not clearly visualize those crystals. The depth of field of the camera proved to be limiting and a different cooling geometry was used, compared to the previous, successful experiments. Analysis to exploit multiple images to improve depth of field and experimental work to understand cooling geometry effects is ongoing. These results will be presented along with advantages and disadvantages of the technique and a discussion of how it might be applied.

Snell, E. H.↗