Section 4.1.6.3. Present results: a summary

Significant and reproducible microgravity experiments have been carried out with a substantial number of model proteins (including lysozyme, thaumatin, canavalin and several plant viruses). The observations in support of microgravity-enhanced crystal growth are primarily of the following nature:

Visual quality and size : The largest dimensions achieved for crystals grown in space were higher than for corresponding crystals grown at 1g. Space-grown crystals were observed to be consistently less marred by cracks, striations, secondary nucleation, visible flaws, inclusions, or aggregate growth. When large numbers of crystals were produced in experiments, morphometric analysis (scoring based on size) of the entire population generally showed a statistically significant tendency toward larger average sizes (e.g. DeLucas et al., 1994; Koszelak et al., 1995; Ng, Lorber et al., 1997).

Maximum resolution and Wilson plots : The first quantitative measurements to support conclusions based on visual inspection were those comparing the maximum resolutions of diffraction patterns from corresponding crystals grown on the ground and in space. A striking improvement of resolution was found for paralbumin, where space-grown crystals diffract to 0.9 Å resolution, but earth-grown crystals are not suitable for diffraction analysis (Declercq et al., 1999).

An analytical procedure for comparing X-ray data is the comparative Wilson plot. Reports have appeared in which the maximum obtainable resolution of X-ray diffraction was greater for crystals grown in space than for equivalent crystals produced on earth. Another product of a Wilson plot is the ratio, over the entire resolution range, of the average intensity to the background scatter, taken in small resolution increments across the entire range. This ratio is, in a way, the peak-to-noise ratio for the measurable X-ray data. Again, as for resolution, the ratio for X-ray diffraction data collected from crystals grown in space was in several cases reported to be greater than for the corresponding earth-grown crystals [e.g. for satellite tobacco mosaic virus (McPherson, 1996) and thaumatin (Ng, Lorber et al., 1997)].

Mosaicity : An additional criterion used to support the enhanced quality of crystals grown in microgravity is the mosaic spread of X-ray diffraction intensities recorded from space- and earth-grown samples. Several reports indicate that for at least some protein crystals (lysozyme, thaumatin), the width and shape of diffraction intensities are improved for crystals grown in microgravity (e.g. Snell et al., 1995; Stojanoff et al., 1996; Ng, Lorber et al., 1997).

Impurity incorporation : Impurities can be incorporated in growing crystals and their partitioning between the crystal and the mother liquor shown (Thomas et al., 1998). Based on theoretical considerations, such partitioning should depend on the presence or absence of convection and, therefore, should be gravity-dependent. This is actually the case as demonstrated with lysozyme, for which the microgravity-grown crystals incorporated 4.5 times less impurity (a lysozyme dimer) than the earth controls (Carter et al., 1999).

Crystallographic structure : In a case study with tetragonal hen egg-white lysozyme crystals, a significant improvement of resolution from 1.6 to 1.35 Å resolution, an average decrease of B factors, and an improved electron density and water structure have been noticed for the space-grown crystals (Carter et al., 1999).

Altogether, the above examples suggest an overall positive effect of microgravity on protein-crystal growth. To date, however, and because of the youth of microgravity science, in particular in its newest developments, it is not possible to make generalizations for all proteins. Even for the same protein, divergent conclusions can be reached; for example, the quality of the X-ray structure of lysozyme was shown to be improved (Carter et al., 1999) or unaffected by microgravity (Vaney et al., 1996). In this case, the contradiction may originate from different levels of impurities present in the protein batches used in the two studies and/or from non-identical growth conditions in different hardware.