Dynamic response of tetragonal lysozyme crystals to changes in relative humidity: implications for post-growth crystal treatments.

The dynamic response of tetragonal lysozyme crystals to dehydration has been characterized in situ using a combination of X-ray topography, high-resolution diffraction line-shape measurements and conventional crystallographic diffraction. For dehydration from 98% relative humidity (r.h.

Triplet-phase measurements using reference-beam X-ray diffraction.

Reference-beam diffraction (RBD) is a recently developed phase-sensitive X-ray diffraction technique that incorporates the principle of multiple-beam diffraction into the standard oscillating-crystal data-collection method [Shen (1998). Phys. Rev.

Enantiomorph determination using inverse reference-beam diffraction images.

It is shown that enantiomorph structures of a noncentrosymmetric crystal can be determined, in the absence of anomalous diffraction signals, by measuring two series of reference-beam oscillation diffraction patterns related by an inverse-beam geometry. The corresponding intensities of the Friedel pairs recorded on the two sets of images exhibit the characteristic three-beam interference effects that provide the unambiguous phase information. The experimental arrangement and the data-analysis procedure are demonstrated through an experimental example on tetragonal lysozyme.

Macromolecular impurities and disorder in protein crystals.

The mechanisms by which macromolecular impurities degrade the diffraction properties of protein crystals have been investigated using X-ray topography, high-resolution diffraction line shape measurements, crystallographic data collection, chemical analysis, and two-photon excitation fluorescence microscopy. Hen egg-white lysozyme crystals grown from solutions containing a structurally unrelated protein (ovotransferrin) and a related protein (turkey egg-white lysozyme) can exhibit significantly broadened mosaicity due to formation of cracks and dislocations but have overall B factors and diffraction resolutions comparable to those of crystals grown from uncontaminated lysozyme. Direct fluorescence imaging of the three-dimensional impurity distribution shows that impurities incorporate with different densities in sectors formed by growth on different crystal faces, and that impurity densities in the crystal core and along boundaries between growth sectors can be much larger than in other parts of the crystal.

X-ray topographic studies of protein crystal perfection and growth.

The effects of solution variations during growth on the perfection of tetragonal lysozyme crystals have been characterized using X-ray topography and high angular and wavevector resolution reciprocal-space scans. X-ray images of crystals grown under nearly uniform conditions show little contrast or evidence of defects, and mosaic widths of these crystals are comparable with those reported for microgravity-grown crystals. Images of crystals for which solution conditions (temperature, pH or salt concentration) are changed after an initial period of uniform growth can show extensive contrast, indicating the presence of disorder.