BUSOC Microgravity Crystallography

Microgravity >> Crystallography

We all are familiar with crystals from rock collections or small molecules, such as salt or sugar. We usually associate them with properties like hard, durable, and pretty. Unfortunately, only the latter is true for protein crystals.

Proteins consist of long macromolecule chains made up from 20 different amino acids. The chains can be several hundred residues long and fold into a 3-dimensional structure. It is therefore quite understandable that protein molecules have irregular shapes and are not ideally suited to be stacked into a periodic lattice, i.e., a crystal. Protein crystals are thus very fragile, soft (think of a cube of jelly instead of a brick) and sensitive to all kind of environmental variations. Protein crystals contain on average 50% solvent, mostly in large channels between the stacked molecules on the crystal. The interactions holding the molecules together are usually weak, hydrogen binds, salt bridges, and hydrophobic interactions, compared to strong covalent or ionic interactions in mineral crystals. This explains the fragility of the crystals, but allows for the possibility of soaking metal solutions (important for phasing) or even large enzyme substrates or inhibitors, into the crystals.

Crystal of parvalbumin grown during the flight STS-83 of the space shuttle, and used for X-ray data collection at atomic resolution (0.91 Å). The scale marker represents 0.5 mm.

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In order to obtain a crystal, the protein molecules must assemble into a periodic lattice. One starts with a solution of the protein with a fairly high concentration (2-50 mg/ml) and adds reagents that reduce the solubility close to spontaneous precipitation. By slow further concentration, and under conditions suitable for the formation of a few nucleation sites, small crystals may start to grow. Often very many conditions have to be tried to succeed. This is usually done by initial screening, followed by a systematic optimization of conditions Crystals should to be a few tenth of a mm in each direction to be useful for diffraction experiments.

The most common setup to grow protein crystals is by the hanging drop technique : A few microliters of protein solution are mixed with an about equal amount of reservoir solution containing the precipitants. A drop of this mixture is put on a glass slide which covers the reservoir. As the protein/precipitant mixture in the drop is less concentrated than the reservoir solution (remember: we mixed the protein solution with the reservior solution about 1:1), water evaporates from the drop into the reservoir. As a result the concentration of both protein and precipitant in the drop slowly increases, and crystals may form. There is a variety of other techniques available such as sitting drops, dialysis buttons, and gel and microbatch techniques. Robots are useful for automatic screening and optimization of crystallization conditions.

Text source: Bernhard Rupp Macromolecular Crystallography

Why Grow Crystals in Microgravity?

Bouyancy Driven Convection

Density-driven convection, or fluid flow, a phenomenon that occurs in normal gravity, is suspected of being a culprit in unusable crystals. Density-driven convection takes place during crystal growth as protein molecules diffuse from surrounding solution and add in an orderly way to the growing crystal lattice. The solution bordering the crystal then contains a lower protein concentration than the remainder of the solution, and therefore, a lower density. This less-dense solution tends to rise, and the denser solution sinks under the influence of gravity, creating eddies next to the crystal. These convective currents are harmful because they alter the orientation of the protein molecules as they add to the crystal lattice, thereby causing disorder of the lattice. This affects the resolution, or clarity, with which a crystallographer can "see" the precise position of each atom occupies in the three-dimensional structure of the protein.

Sedimentation

Another adverse effect of gravity on growing crystal is sedimentation. Crystals drift to the bottom of a drop of the solution when they have grown to a mass larger than can be supported by suspension in the drop. When this happens, partially formed crystals fall on top of one another and continue growing into each other. Since X-ray diffraction analysis requires single crystals, sedimentation renders potentially high-quality crystals unusable for data collection.

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