Average e3 mosacity versus cell volume (relative to RT) of tetragonal thermolysin crystals, comparing plunge cooling into liquid nitrogen at 77 K with gas stream cooling at 100 K. In each case, cryosolvents of 50%(w/w) cryoprotective agent with water were used. The fractional change in specific volume of the cryosolvent with cooling between RT and 77 K is shown in parentheses. Greater contracting cryosolvents produce both smaller cell volumes and larger mosaicities (Juers et al., 2018). Plunge cooling yields more disorder and slightly larger cell volumes than gas stream cooling. In all cases, no ice was observed in the diffraction pattern. For each data point, 3–11 crystals were tested, and the error bars are standard errors. Mosaicities and cell volumes are based on pre-experiments (see Methods). The plunge cooling procedure used is protocol (3) in Section 2.2: normal plunge cooling using a dewar of liquid nitrogen and moving quickly through a 1–3 cm-thick cold gas layer into the liquid nitrogen below.