Pinpointing protein structures over broad temperature range using hydrophobic protection

Protein interactions are dynamic processes influenced by chemical and physical variables. While variable temperatures play an important role in understanding biological processes, their use in protein crystallography has been limited due to the loss of diffraction power. Here, we report the selection of a cryo-protectant that enables X-ray diffraction data collection across a continuum of temperatures, from cryogenic to room temperature. Although several hydrophobic materials effectively preserved samples at 100 K, only a few compounds allowed data collection at 300 K. We identified a hydrophobic grease suitable for both home-source and synchrotron applications, supporting ultrafast and slow diffraction, with exposure times as long as 60 seconds per image at atomic resolution. Data collected between 100 K and 300 K showed no significant effect of exposure time (ranging from 70 msec to 1 second) but revealed an exponential temperature dependence on the overall B-factor. These findings highlight the importance of hydrophobic grease in protecting against environmental variables, enabling optimized data collection across an ultra-wide temperature range.