Preparation of crystals of T7 RNA polymerase suitable for high-resolution X-ray structure analysis

The crystallization of bacteriophage T7 RNA polymerase is described. A number of features of the crystallization methodology are worthy of note: (1) Preparation of crystals suitable for X-ray analysis depended on removal of oligomeric forms of the enzyme which formed during purification and were not detectable by denaturing gel electrophoreris. (2) By increasing the protein supersaturation, changes in the relative interfacial growth rates were induced, resulting in increases in crystal thickness and diffraction to higher resolution. (3) The stability of the crystalline versus amorphous phase of the solid protein was shifted by the presence of glycerol in the mother liquor: crystallization was dependent on the presence of at least 15% glycerol. The high density and viscosity of glycerol mother liquors reduced convective and diffusive flow and eliminated crystal sedimentation. The implications and possible mechanisms of the glycerol effect on crystallization are discussed and the generality and extension of this effect is suggested.