Cryocrystallography in capillaries: critical glycerol concentrations and cooling rates

Abstract

Capillary tubes have many advantages over multi-well plates for macromolecular crystal growth and handling, including the possibility ofin situstructure determination. To obtain complete high-resolution X-ray data sets, cryopreservation protocols must be developed to prevent crystalline ice formation and preserve macromolecular crystal order. The minimum glycerol concentrations required to vitrify aqueous solutions during plunging into liquid nitrogen and liquid propane have been determined for capillary diameters from 3.3 mm to 150 µm. For the smallest diameter, the required glycerol concentrations are 30%(w/v) in nitrogen and 20%(w/v) in propane, corresponding to cooling rates of ∼800 and ∼7000 K s−1, respectively. These concentrations are much larger than are required in current best practice using crystals in loops or on microfabricated mounts. In additon, the relation between the minimum cooling rate for vitrification and glycerol concentration has been estimated; this relation is of fundamental importance in developing rational cryopreservation protocols.