A fast, simple and robust protocol for growing crystals in the lipidic cubic phase

Abstract

A simple and inexpensive protocol for producing crystals in the sticky and viscous mesophase used for membrane protein crystallization by the in meso method is described. It provides crystals that appear within 15-30 min of setup at 293 K. The protocol gives the experimenter a convenient way of gaining familiarity and a level of comfort with the lipidic cubic mesophase, which can be daunting as a material when first encountered. Having used the protocol to produce crystals of the test protein, lysozyme, the experimenter can proceed with confidence to apply the method to more valuable membrane (and soluble) protein targets. The glass sandwich plates prepared using this robust protocol can further be used to practice harvesting and snap-cooling of in meso-grown crystals, to explore diffraction data collection with mesophase-embedded crystals, and for an assortment of quality control and calibration applications when used in combination with a crystallization robot.

Figure 1

Steps involved in manually setting up in meso crystallization trials with lysozyme following a protocol that produces crystals within 30 min at 293 K. An open-access video of the protocol can be viewed online (Caffrey & Porter, 2010 ▶).

Figure 2

Crystallization of lysozyme at 293 K in the lipidic cubic phase prepared with monoolein as the hosting lipid. (a) A view of a suitably positioned 200 nl mesophase (m) bolus surrounded by 1 µl of precipitant (p) solution in a 6 mm-diameter well (w). (b) A close-up view in normal light of the mesophase immediately upon setup. The edge (e) of the mesophase bolus is marked with an arrow. (c) An expanded view in normal light of the mesophase 30 min after setup. Small crystals are obvious as dark flecks around the perimeter of the bolus. (d) As in (c), viewed between crossed polarizers. (e) An expanded view of the mesophase in normal light 60 min after setup. Crystals are clearly visible as dark flecks around the perimeter of the bolus. (f) As in (e), viewed between crossed polarizers. Crystals are apparent as bright flecks on a dark background. (g) An expanded view in normal light of crystals growing in the lipidic mesophase 85 min after setup.