Protein conformation and crystal packing. (a) Dependence of the LT hinge-bending angle between the N-terminal and C-terminal domains of thermolysin on cryosolution contraction. The reference state is tetragonal thermolysin with methanol as a cryoprotectant. More negative values correspond to more `closed' conformations. Domain definitions from Holland et al. (1992) were used. The RT values for tetragonal thermolysin are −1.9 and −1.8° (for MPD and xylose; ΔTsol = −0.085 and −0.030, respectively). This RT effect accounts for ∼30% of the dependence of the tetragonal thermolysin hinge-bending angle on cryosolvent contraction, suggesting that the remainder is owing to differential contraction. RT values for hexagonal thermolysin are −6.0 and −5.9° (for 50% DMF and 50% xylose; ΔTsol = −0.105 and −0.030, respectively), over the region of little change in the hexagonal thermolysin hinge-bending angle. (b) Dependence of structural difference (Cα r.m.s.d.) on unit-cell difference for tetragonal thermolysin. If the cell volumes are similar, two LT structures can be as similar as two RT structures. However, increasing cell-volume difference correlates with larger LT structural difference, so that two LT structures can be as different as an LT and an RT structure. (c) The relationship between crystal contacts and solvent contraction. Crystal contacts were calculated using a uniform 4.0 Å centre-to-centre distance cutoff for all atoms using EdPDB (Zhang & Matthews, 1995). All LT structures were compared with the 50%(w/w) RT xylose soaks. In most cases, cooling increases the number of crystal contacts. For the thermolysins, greater solvent contraction increases the number of crystal contacts relative to room temperature, while this trend is not obvious for orthorhombic trypsin. All LT structures with positive values of ΔTsol showed some ice formation. Note that the highest ratio for trypsin occurred with the greatest cell reduction at 20% xylose with ice formation. Other levels of stringency for calculating contacts showed qualitatively similar results (Supplementary Fig. S3).