Macromolecular impurities and disorder in protein crystals

The mechanisms by which macromolecular impurities degrade the diffraction p roperties of protein crystals have been investigated using X-ray topography , high-resolution diffraction line shape measurements, crystallographic dat a collection, chemical analysis, and two-photon excitation fluorescence mic roscopy. Hen egg-white lysozyme crystals grown from solutions containing a structurally unrelated protein (ovotransferrin) and a related protein (turk ey egg-white lysozyme) can exhibit significantly broadened mosaicity due to formation of cracks and dislocations but have overall B factors and diffra ction resolutions comparable to those of crystals grown from uncontaminated lysozyme. Direct fluorescence imaging of the three-dimensional impurity di stribution shows that impurities incorporate with different densities in se ctors formed by growth on different crystal faces, and that impurity densit ies in the crystal core and along boundaries between growth sectors can be much larger than in other parts of the crystal. These nonuniformities creat e stresses that drive formation of the defects responsible for the mosaic b roadening. Our results provide a rationale for the use of seeding to obtain high-quality crystals from heavily contaminated solutions and have implica tions for the use of crystallization for protein purification. (C) 1999 Wil ey Liss, Inc.