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Cryo-cooling has been nearly universally adopted to mitigate X-ray damage and facilitate crystal handling in protein X-ray crystallography. However, cryo X-ray crystallographic data provide an incomplete window into the ensemble of conformations that is at the heart of protein function and energetics. Room-temperature (RT) X-ray crystallography provides accurate ensemble information, and recent developments allow conformational heterogeneity (the experimental manifestation of ensembles) to be extracted from single-crystal data. Nevertheless, high sensitivity to X-ray damage at RT raises concerns about data reliability. To systematically address this critical issue, increasingly X-ray-damaged high-resolution data sets (1.02–1.52 Å resolution) were obtained from single proteinase K, thaumatin and lysozyme crystals at RT (277 K). In each case a modest increase in conformational heterogeneity with X-ray damage was observed. Merging data with different extents of damage (as is typically carried out) had negligible effects on conformational heterogeneity until the overall diffraction intensity decayed to ∼70% of its initial value. These effects were compared with X-ray damage effects in cryo-cooled crystals by carrying out an analogous analysis of increasingly damaged proteinase K cryo data sets (0.9–1.16 Å resolution). X-ray damage-associated heterogeneity changes were found that were not observed at RT. This property renders it difficult to distinguish real from artefactual conformations and to determine the conformational response to changes in temperature. The ability to acquire reliable heterogeneity information from single crystals at RT, together with recent advances in RT data collection at accessible synchrotron beamlines, provides a strong motivation for the widespread adoption of RT X-ray crystallography to obtain conformational ensemble information. 

The mechanisms that underly the adaptation of enzyme activities and stabilities to temperature are fundamental to our understanding of molecular evolution and how enzymes work. Here, we investigate the molecular and evolutionary mechanisms of enzyme temperature adaption, combining deep mechanistic studies with comprehensive sequence analyses of thousands of enzymes. We show that temperature adaptation in ketosteroid isomerase (KSI) arises primarily from one residue change with limited, local epistasis, and we establish the underlying physical mechanisms. This residue change occurs in diverse KSI backgrounds, suggesting parallel adaptation to temperature. We identify residues associated with organismal growth temperature across 1005 diverse bacterial enzyme families, suggesting widespread parallel adaptation to temperature. We assess the residue properties, molecular interactions, and interaction networks that appear to underly temperature adaptation.