Flash-cooling and annealing of macromolecular crystals have been investigated using in situ X-ray imaging, diffraction-peak lineshape measurements and conventional crystallographic diffraction. The dominant mechanisms by which¯ash-cooling creates disorder are suggested and a ®xed-temperature annealing protocol for reducing this disorder is demonstrated that should be more reliable and¯exible than existing protocols. Flash-cooling tetragonal lysozyme crystals degrades diffraction resolution and broadens the distributions of lattice orientations (mosaicity) and lattice spacings. The diffraction resolution strongly correlates with the width of the latticespacing distribution. Annealing at ®xed temperatures of 253 and 233 K consistently reduces the lattice-spacing spread and improves the resolution for annealing times up to $30 s. X-ray images show that this improvement arises from the formation of well ordered domains with characteristic sizes >10 mm and narrower mosaicities than the crystal as a whole. Flash-cooled triclinic crystals of lysozyme, which have a smaller water content than the tetragonal form, diffract to higher resolution with smaller mosaicities and exhibit pronounced ordered domain structure even before annealing. It is suggested that differential thermal expansion of the protein lattice and solvent may be the primary cause of¯ash-cooling-induced disorder. Mechanisms by which annealing at T << 273 K reduce this disorder are discussed.

Three problems involving heat transfer from a protein crystal to a cooling agent are analyzed: flash-cooling in a cold nitrogen- or helium-gas stream, plunge-cooling into liquid nitrogen, propane or ethane and crystal heating in a cold gas stream owing to X-ray absorption. Heat transfer occurs by conduction inside the crystal and by convection from the crystal's outer surface to the cooling fluid. For flash-cooling in cold gas streams, heat transfer is limited by the rate of external convection; internal temperature gradients and crystal strains during cooling are very small. Helium gas provides only a threefold improvement in cooling rates relative to nitrogen because its much larger thermal conductivity is offset by its larger kinematic viscosity. Characteristic cooling times vary with crystal size L as L(3/2) and theoretical estimates of these times are consistent with experiments. Plunge-cooling into liquid cryogens, which can give much smaller convective thermal resistances provided that surface boiling is eliminated, can increase cooling rates by more than an order of magnitude. However, the internal conduction resistance is no longer negligible, producing much larger internal temperature gradients and strains that may damage larger crystals. Based on this analysis, factors affecting the success of flash-cooling experiments can be ordered from most to least important as follows: (1) crystal solvent content and solvent composition, (2) crystal size and shape, (3) amount of residual liquid around the crystal, (4) cooling method (liquid plunge versus gas stream), (5) choice of gas/liquid and (6) relative speed between cooling fluid and crystal. Crystal heating by X-ray absorption on present high-flux beamlines should be small. For a fixed flux and illuminated area, heating can be reduced by using crystals with areas normal to the beam that are much larger than the beam area.

Quasilongitudinal sound velocities and the second-order elastic moduli of tetragonal hen egg-white lysozyme crystals were determined as a function of relative humidity (RH) by Brillouin scattering. In hydrated crystals the measured sound velocities in the [110] plane vary between 2.12 +/- 0.03 km/s along the [001] direction and 2.31 +/- 0.08 km/s along the [110] direction. Dehydration from 98% to 67% RH increases the sound velocities and decreases the velocity anisotropy in (110) from 8.2% to 2.0%. A discontinuity in velocity and an inversion of the anisotropy is observed with increasing dehydration providing support for the existence of a structural transition below 88% RH. Brillouin linewidths can be described by a mechanical model in which the phonon is coupled to a relaxation mode of hydration water with a single relaxation time of 55 +/- 5 ps. At equilibrium hydration (98% RH) the longitudinal moduli C(11) + C(12) + 2C(66) = 12.81 +/- 0.08 GPa, C(11) = 5.49 +/- 0.03 GPa, and C(33) = 5.48 +/- 0.05 GPa were directly determined. Inversion of the measured sound velocities in the [110] plane constrains the combination C(44) + (1/2)C(13) to 2.99 +/- 0.05 GPa. Further constraints on the elastic tensor are obtained by combining the Brillouin quasilongitudinal results with axial compressibilities determined from high-pressure x-ray diffraction. We constrain the adiabatic bulk modulus to the range 2.7-5.3 GPa.

The dynamic response of tetragonal lysozyme crystals to dehydration has been characterized in situ using a combination of X-ray topography, high-resolution diffraction lineshape measurements and conventional crystallographic diffraction. For dehydration from 98% relative humidity (r.h.) to above 89%, mosaicity and diffraction resolution show little change and X-ray topographs remain featureless. Lattice constants decrease rapidly but the lattice-constant distribution within the crystal remains very narrow, indicating that water concentration gradients remain very small. Near 88% r.h., the c-axis lattice parameter decreases abruptly, the steady-state mosaicity and diffraction resolution degrade sharply and topographs develop extensive contrast. This transformation exhibits metastability and hysteresis. At ®xed r.h. < 88% it is irreversible, but the original order can be almost completely restored by rehydration. These results suggest that this transformation is a ®rst-order structural transition involving an abrupt loss of crystal water. The front between transformed and untransformed regions may propagate inward from the crystal surface and the resulting stresses along the front may degrade mosaicity. Differences in crystal size, shape and initial perfection may produce the observed variations in degradation timescale. Consequently, the success of more general post-growth treatments may often involve identifying procedures that either avoid lattice transitions, minimize disorder created during such transitions or maintain the lattice in an ordered metastable state.

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. These nonuniformities create stresses that drive formation of the defects responsible for the mosaic broadening. Our results provide a rationale for the use of seeding to obtain high-quality crystals from heavily contaminated solutions and have implications for the use of crystallization for protein purification. Proteins 1999;36:270-281.