Crystallization of Old Yellow Enzyme illustrates an effective strategy for increasing protein crystal size

Old Yellow Enzyme (OYE) was the first flavoenzyme discovered, and has been widely used as a model flavoenzyme. Three crystal forms have been grown from natural protease-nicked OYE and one has been grown from intact recombinant OYE. The recombinant OYE crystals are best suited for structure determination and grow from a solution containing polyethylene glycol and MgCl2. They belong to space group P4(3)2(1)2 with unit cell dimensions a = b = 142.88 A, c = 43.01 A and have a single OYE chain per asymmetric unit. In order to optimize the production of large single crystals, we have carried out a systematic study of crystal size versus drop volume. Crystallographic lore holds that the volume of the medium in which the crystals are grown should have a significant effect on the final size of the crystals produced. However, a systematic investigation of the degree to which the volume of the crystallization sample affects the final size of the crystals has not been reported. In order to investigate this phenomenon, drops were set up in both hanging and sandwich geometry at a variety of drop volumes, but otherwise identical crystallization conditions. A 60-fold increase in drop volume produced a 730-fold increase in crystal volume, and a simultaneous increase in the effective diffraction limit of the crystals from near 2.5 A to well beyond 2.0 A resolution. The dramatic crystal volume increase appears to be due both to the increased amount of protein and the slower equilibration rates associated with larger drops. Our results also emphasize that the quality of diffraction data may often be limited by crystal size rather than intrinsic crystal order even in the case of reasonably sized (200 to 400 microns) crystals.