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

Abstract

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.

Obtaining anomalous and ensemble information from protein crystals from 220 K up to physiological temperatures

“…Crystal lifetimes, measured as 'the time until the I/I 0 ratio decreases below 0.7', increase with cooling. Based on work on X-ray damage at cryotemperatures (Owen et al, 2006) and at RT (Yabukarski, Doukov, Mokhtari et al, 2022;de la Mora et al, 2020), this value of I/I 0 was proposed as a limit for intensity decay due to X-ray damage. For lysozyme at 293 K it took 62.4 s (1560 ), at 273 K it took 72 s (1800 ) and at 220 K it took 288 s (2880 ).…”

“…The data-collection approach used by Greisman and coworkers is largely based on traditional methods for anomalous data collection using large crystals inside MicroRT sleeves (MiTeGen), 5 Â 30 mm (FWHM) helical data collection at 6.55 keV energy at a synchrotron and 26-35-fold redundant data sets. Here, we report an approach based on a recently developed method for data collection at synchrotrons across temperature which differs from traditional data-collection methods and has been optimized for rapid data collection with minimized X-ray damage (Doukov et al, 2020;Yabukarski, Doukov, Mokhtari et al, 2022). Given the recent emphasis on obtaining conformational ensemble information to understand function (Henzler-Wildman et al, 2007;Klinman, 2015;Yabukarski, Doukov, Pinney et al, 2022), RT and physiological temperature (PT) X-ray diffraction data can be used to obtain conformational heterogeneity information for proteins (van den Bedem et al, 2009;Riley et al, 2021;Lang et al, 2010;Burnley et al, 2012;Ploscariu et al, 2021), with the conformational heterogeneity being the experimental manifestation of the ensemble nature of proteins .…”

Introduction to the virtual thematic issue on room-temperature biological crystallography

“…The theme of ligand-binding discrepancies between cryo-cooled and physiological temperature structures is discussed by Huang and co-workers by looking at the interaction of TL00150, a 175.15 Da fragment, with endothiapepsin using a 'temperature-resolved' approach (Huang et al, 2022). Some aspects of radiation damage are considered by Yabukarski and colleagues who employed a panel of well known model systems, to show that radiation damage only modestly increases conformational heterogeneity at RT, suggesting that dynamic information can be obtained from single crystals at nearphysiological temperature (Yabukarski et al, 2022). Ba ˚th and co-authors employed lipidic cubic phase (LCP) microcrystallization applied to the photosynthetic reaction centre of Blastochloris viridis (Ba ˚th et al, 2022).…”

Probing ligand binding of endothiapepsin by `temperature-resolved' macromolecular crystallography

“…A guiding limit of 380 kGy has been proposed (de la Mora et al, 2020). More importantly, studies have suggested that specific and global radiation damage are less decoupled at room temperature than at cryogenic temperatures, where specific damage is found to occur more quickly (Gotthard et al, 2019), which makes it possible to measure protein conformational heterogeneity reliably (Russi et al, 2017;Yabukarski et al, 2022).…”