Conformational variation of proteins at room temperature is not dominated by radiation damage

Abstract

Protein crystallography data collection at synchrotrons is routinely carried out at cryogenic temperatures to mitigate radiation damage. Although damage still takes place at 100 K and below, the immobilization of free radicals increases the lifetime of the crystals by approximately 100-fold. Recent studies have shown that flash-cooling decreases the heterogeneity of the conformational ensemble and can hide important functional mechanisms from observation. These discoveries have motivated increasing numbers of experiments to be carried out at room temperature. However, the trade-offs between increased risk of radiation damage and increased observation of alternative conformations at room temperature relative to cryogenic temperature have not been examined. A considerable amount of effort has previously been spent studying radiation damage at cryo-temperatures, but the relevance of these studies to room temperature diffraction is not well understood. Here, the effects of radiation damage on the conformational landscapes of three different proteins (T. danielli thaumatin, hen egg-white lysozyme and human cyclophilin A) at room (278 K) and cryogenic (100 K) temperatures are investigated. Increasingly damaged datasets were collected at each temperature, up to a maximum dose of the order of 10⁷ Gy at 100 K and 10⁵ Gy at 278 K. Although it was not possible to discern a clear trend between damage and multiple conformations at either temperature, it was observed that disorder, monitored by B-factor-dependent crystallographic order parameters, increased with higher absorbed dose for the three proteins at 100 K. At 278 K, however, the total increase in this disorder was only statistically significant for thaumatin. A correlation between specific radiation damage affecting side chains and the amount of disorder was not observed. This analysis suggests that elevated conformational heterogeneity in crystal structures at room temperature is observed despite radiation damage, and not as a result thereof.

Keywords: conformational dynamics; cryo-temperature; radiation damage; room temperature.

Figure 1

Example of buried isoleucine (I56 in CypA at 278 K) in a 2F _o − F _c map contoured at 1.0σ and 0.3σ. At the lowest absorbed dose (a) only one rotamer is modelled. At a higher dose (b) an alternate rotamer occupying the same density is fitted without visible changes in the map.

Figure 2

Di­sulfide bond C121—C193 in thaumatin at the highest absorbed dose. At 100 K (a), one of the cysteines could be modelled in an alternate conformation, while negative density, contoured at 2.5σ between the bonded sulfurs, indicates that photoreduction has taken place. At 278 K (b), the negative density is not visible at the same contour level and the map does not support modelling of a second conformation.

Figure 3

2F _o − F _c electron density maps showing the CypA F113 side chain contoured at 1σ (blue) and 0.3σ (light blue) for increasingly damaged data. The least damaged model (in yellow) is shown together with the more damaged models for reference. The DWD for each model was (a) 0.016 MGy, (b) 0.038 MGy, (c) 0.06 MGy and (d) 0.124 MGy. As radiation damage progresses, the electron density for the minor conformer becomes less defined and the refined position of the alternate side chain moves closer to the major conformer.

Figure 4

Average side-chain order parameters as a function of DWD for HEWL (a), thaumatin (b) and CypA (c) at 100 and 278 K.

Figure 5

Order parameters as a function of DWD for the CypA residues R55 (slope = −0.199; p = 0.513), M61 (slope = −0.153; p = 0.373), S99 (slope = 0.643; p = 0.161) and F113 (slope = −0.579; p = 0.201) at 278 K.