Crystals of insulin grown in microgravity on Space Shuttle Mission STS-95 were extremely well ordered and unusually large (many >2 mm). The physical characteristics of six microgravity and six earthgrown crystals were examined by X-ray analysis employing super®ne 9 slicing and unfocused synchrotron radiation. This experimental setup allowed hundreds of re¯ections to be precisely examined from each crystal in a short period of time. The microgravity crystals were on average 34 times larger, had sevenfold lower mosaicity, had 54-fold higher re¯ection peak heights and diffracted to signi®cantly higher resolution than their earth-grown counterparts. A single mosaic domain model could account for the observed re¯ection pro®les in microgravity crystals, whereas data from earth crystals required a model with multiple mosaic domains. This statistically signi®cant and unbiased characterization indicates that the microgravity environment was useful for the improvement of crystal growth and the resultant diffraction quality in insulin crystals and may be similarly useful for macromolecular crystals in general.

Typical measurements of macromolecular crystal mosaicity are dominated by the characteristics of the X‐ray beam and as a result the mosaicity value given during data processing can be an artifact of the instrumentation rather than the sample. For physical characterization of crystals, an experimental system and software have been developed to simultaneously measure the diffraction resolution and mosaic spread of macromolecular crystals. The contributions of the X‐ray beam to the reflection angular widths were minimized by using a highly parallel, highly monochromatic synchrotron source. Hundreds of reflection profiles over a wide resolution range were rapidly measured using a charge‐coupled device (CCD) area detector in combination with superfine ϕ‐slicing data collection. The Lorentz effect and beam contributions were evaluated and deconvoluted from the recorded data. Data collection and processing is described. From 1° of superfine ϕ‐­slice data collected on a crystal of manganese superoxide dismutase, the mosaicities of 260 reflections were measured. The average mosaicity was 0.0101° (s.d. 0.0035°) measured as the full‐width at half‐maximum (FWHM) and ranged from 0.0011 to 0.0188°. Each reflection profile was individually fitted with two Gaussian profiles, with the first Gaussian contributing 55% (s.d. 9%) and the second contributing 35% (s.d. 9%) of the reflection. On average, the deconvoluted width of the first Gaussian was 0.0054° (s.d. 0.0015°) and the second was 0.0061° (s.d. 0.0023°). The mosaicity of the crystal was anisotropic, with FWHM values of 0.0068, 0.0140 and 0.0046° along the a,  b and c axes, respectively. The anisotropic mosaicity analysis indicates that the crystal is most perfect in the direction that corresponds to the favored growth direction of the crystal.

Chicken egg-white lysozyme (CEWL) crystals were grown in microgravity and on the ground in the presence of various amounts of a naturally occurring lysozyme dimer impurity. No significant favorable differences in impurity incorporation between microgravity and ground crystal samples were observed. At low impurity concentration the microgravity crystals preferentially incorporated the dimer. The presence of the dimer in the crystallization solutions in microgravity reduced crystal size, increased mosaicity, and reduced the signal-to-noise ratio of the X-ray data. Microgravity samples proved more sensitive to impurity. Accurate indexing of the reflections proved critical to the X-ray analysis. The largest crystals with the best X-ray diffraction properties were grown from pure solution in microgravity.

For a normal periodic crystal, the X‐ray diffraction pattern can be described by an orientation matrix and a set of three integers that indicate the reciprocal lattice points. Those integers determine the spacing along the reciprocal lattice directions. In aperiodic crystals, the diffraction pattern is modulated and the standard periodic main reflections are surrounded by satellite reflections. The successful indexing and refinement of the main unit cell and q vector using TWINSOLVE, developed by Svensson [(2003). Lund University, Sweden], are reported here for an incommensurately modulated, aperiodic crystal of a profilin:actin complex. The indexing showed that the modulation is along the b direction in the crystal, which corresponds to an `actin ribbon' formed by the crystal lattice. Interestingly, the transition to the aperiodic state was shown to be reversible and the diffraction pattern returned to the periodic state during data collection. It is likely that the protein underwent a conformational change that affected the neighbouring profilin:actin molecules in such a way as to produce the observed modulation in the diffraction pattern. Future work will aim to trap the incommensurately modulated crystal state, for example using cryocooling or chemical crosslinking, thus allowing complete X‐ray data to be collected.