Use of cryoprotectants in combination with immiscible oils for flash cooling macromolecular crystals

Two standard methods have generally been used to prevent destructive ice formation when flash cooling macromolecular crystals for cryocrystallography: (i) permeating the crystal lattice with a cryoprotectant, or (ii) replacing the external liquid around the crystal with an immiscible oil. In two cases where both of the standard methods failed, we obtained satisfactory results with a two-step procedure that combined the use of immiscible oil with diffusable cryoprotectant. The procedure enabled substantially lower concentrations of cryoprotectant to be used. Since many macromolecular crystals are susceptible to high concentrations of cryoprotectant and successful flash cooling is often a trade-off between damage from ice formation and damage from cryoprotectant, this combinatory procedure may find general application.