Dark progression reveals slow timescales for radiation damage between T = 180 and 240 K

Abstract

Can radiation damage to protein crystals be `outrun' by collecting a structural data set before damage is manifested? Recent experiments using ultra-intense pulses from a free-electron laser show that the answer is yes. Here, evidence is presented that significant reductions in global damage at temperatures above 200 K may be possible using conventional X-ray sources and current or soon-to-be available detectors. Specifically, `dark progression' (an increase in damage with time after the X-rays have been turned off) was observed at temperatures between 180 and 240 K and on timescales from 200 to 1200 s. This allowed estimation of the temperature-dependent timescale for damage. The rate of dark progression is consistent with an Arrhenius law with an activation energy of 14 kJ mol(-1). This is comparable to the activation energy for the solvent-coupled diffusive damage processes responsible for the rapid increase in radiation sensitivity as crystals are warmed above the glass transition near 200 K. Analysis suggests that at T = 300 K data-collection times of the order of 1 s (and longer at lower temperatures) may allow significant reductions in global radiation damage, facilitating structure solution on crystals with liquid solvent. No dark progression was observed below T = 180 K, indicating that no important damage process is slowed through this timescale window in this temperature range.

Figure 1

Schematic representation of global radiation sensitivity versus inverse temperature for thaumatin crystals. Different damage mechanisms have been suggested to dominate in each of the three distinct temperature regimes.

Figure 2

Probing damage timescales with X-rays. (a) An ‘impulse-response’ measurement. A very short, very intense pulse delivers a large dose and the evolution of damage is then monitored using short weaker pulses. (b) Maximum available X-ray flux density (dose rate) and minimum doses required to assess damage mean that both the damaging and probe pulses must have finite widths. These widths determine the time resolution of the experiment. (c) Schematic representation of the dosing sequence used in the present experiments.

Figure 3

An interrupted dose curve of relative B factor versus dose acquired at T = 200 K. The time between data points was 40 s. The dark-interval times at each interruption were 240 and 660 s for the first and second interruptions, respectively. The solid line is a fit to the model discussed in §5.3 with α/β = 2 and τ = 240 s. The vertical distance between the dotted line and the data collected immediately after the dark interval determines the amount of dark progression.

Figure 4

Interrupted dose curves at temperatures between 25 and 300 K where no dark progression was observed. The time between data points was 40 s. The dark-interval times at each interruption (indicated by the arrows) were 840, 1080, 1320, 1320, 1320, 1080, 600 and 600 s at T = 25, 50, 80, 100, 130 150, 255 and 300 K, respectively. At each arrow, a jump in the curve would indicate the presence of dark progression (as in Fig. 3 ▶), so the absence of jumps indicates that no additional damage was manifested during the dark-interval time. Crystal sensitivity to dose and thus the slope of relative B factor versus dose varies strongly with temperature. The doses for each curve in Fig. 4 ▶ have been normalized to give the same slope and the normalization factor (sensitivity) versus temperature is shown in the inset. The curves are also vertically offset for clarity.

Figure 5

Dark progression (rise in relative B factor) versus dark-interval time at temperatures between 180 and 240 K obtained from data such as those shown in Fig. 3 ▶. Each time the dose curve was interrupted, the B factor rose by some amount while the X-rays were off, and that rise is shown here as a function of the the dark interval. At each temperature, larger dark intervals produce more damage. Evidence for saturation of damage with dark-interval time is seen at 240 K. The straight lines show linear fits to the initial (<600 s) slope of the data at each temperature; these slopes are shown in Fig. 6 ▶.

Figure 6

Arrhenius plot of dark-progression rate versus inverse temperature, as determined from the slopes of the linear fits in Fig. 5 ▶. Between 240 and 180 K the data are consistent with a single thermally activated process with an activation energy of 14 ± 1 kJ mol⁻¹, indicated by the solid line fit.

Figure 7

Schematic illustration of how global radiation damage may evolve with time in response to a very short intense X-ray pulse delivered at t = 0. Solid lines show the damage response assuming two exponential processes: a fast process with a temperature-independent timescale of 1 ns and a slow process whose timescale is temperature-activated. Dashed lines show the damage response assuming that the slow process has a broad distribution of relaxation times characteristic of glassy or dis­ordered systems. The blue (bottom right) line shows the response at T = 100, where the slow temperature-activated processes have frozen out.