Time Resolved Ice Formation in Protein Crystals

A combination of solvent nanoconfinement, fast cooling, and fast data collection enables ice-free protein crystallography between 180 and 260 K, even in crystals having large solvent cavities and large bulk-like solvent fractions. The time evolution of diffraction properties was measured for fast-cooled crystals of apoferritin (63% solvent content, 33.9 Å solvent cavities) and of thaumatin (58% solvent content, 12.4 Å solvent cavities) soaked in solutions containing between 0% and 40% w/v glycerol. While ice formation in external solvent occurs rapidly below 260 K, internal solvent is suppressed and occurs stochastically. Removing external solvent reliably produces ice-free diffraction at between 180 K and 260 K for at least seconds, sufficient for collection of complete data sets. At 200 to 240 K, crystal mosaicities are much smaller than those obtained at 100 K with lowest mosaicities from cryoprotectant-free crystals. While the internal solvent remains liquid, solvent transport within the crystal and structural relaxations of the protein molecules and of the crystal lattice can occur. These results make variable temperature crystallography viable for protein crystals with large solvent contents, and with weakly bound ligands that may be displaced by cryorpotectants.