Protein crystal movements and fluid flows during microgravity growth

The growth of protein crystals suitable for X–ray crystal structure analysis is an important topic. The methods of protein crystal growth are under increasing study whereby different methods are being compared via diagnostic monitoring including charge coupled device (CCD) video and interferometry. The quality (perfection) of protein crystals is now being evaluated by mosaicity analysis (rocking curves) and X–ray topographic images as well as the diffraction resolution limit and overall data quality. Choice of a liquid—liquid linear crystal–growth geometry and microgravity can yield a spatial stability of growing crystals and fluid, as seen in protein crystallization experiments on the uncrewed platform EURECA. A similar geometry used within the Advanced Protein Crystallization Facility (APCF) onboard the crewed shuttle missions SpaceHab–01 and IML–2, however, has shown by CCD video some lysozyme crystal movement through the mother liquor. Moreover, spurts and lulls of growth of a stationary lysozyme protein crystal that was probably fixed to the crystal–growth reactor wall suggests g–jitter stimulated movement of fluid on IML–2, thus transporting new protein to the growing crystal faces. In yet another study, use of a hanging drop vapour diffusion geometry on the IML–2 shuttle mission showed, again via CCD video monitoring, growing apocrustacyanin C1 protein crystals executing near cyclic movement, reminiscent of Marangoni convection flow of fluid, the crystals serving as ‘markers’ of the fluid flow. These observations demonstrated that the use of vapour diffusion geometry did not yield spatially stable crystal position or fluid conditions for a solely protein diffusive regime to be realized. Indeed mosaicity evaluation of those vapour diffusion–grown apocrustacyanin C1 crystals showed inconsistent protein crystal quality, although the best crystal studied was microgravity grown. In general, realizing perfect conditions for protein crystal growth, of absence of movement of crystal or fluid, requires not only the correct choice of geometry but also the avoidance of low–frequency (≲5Hz) g–jitters. A review is given here of existing results and experience over several microgravity missions. Some comment is given on gel protein crystal growth in attempts to ‘mimic’ the benefits of microgravity on Earth. Finally, the recent new results from our experiments on the shuttle mission LMS are described. These results include CCD video as well as interferometry during the mission, followed, on return to Earth, by reciprocal space mapping at the NSLS, Brookhaven and full X–ray data collection on LMS and Earth control lysozyme crystals. Diffraction data recorded from LMS and ground control apocrustacyanin C1 crystals are also described.