Evaluating the impact of X-ray damage on conformational heterogeneity in room-temperature (277   K) and cryo-cooled protein crystals

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 re...
Source: Acta Crystallographica Section D - Category: Biochemistry Authors: Tags: protein X-ray crystallography X-ray damage room temperature conformational ensembles hydrogen bonds research papers Source Type: research