Lifetimes and spatio-temporal response of protein crystals in intense X-ray microbeams

Abstract

Serial synchrotron-based crystallography using intense microfocused X-ray beams, fast-framing detectors and protein microcrystals held at 300 K promises to expand the range of accessible structural targets and to increase overall structure-pipeline throughputs. To explore the nature and consequences of X-ray radiation damage under microbeam illumination, the time-, dose- and temperature-dependent evolution of crystal diffraction have been measured with maximum dose rates of 50 MGy s^-1. At all temperatures and dose rates, the integrated diffraction intensity for a fixed crystal orientation shows non-exponential decays with dose. Non-exponential decays are a consequence of non-uniform illumination and the resulting spatial evolution of diffracted intensity within the illuminated crystal volume. To quantify radiation-damage lifetimes and the damage state of diffracting crystal regions, a revised diffraction-weighted dose (DWD) is defined and it is shown that for Gaussian beams the DWD becomes nearly independent of actual dose at large doses. An apparent delayed onset of radiation damage seen in some intensity-dose curves is in fact a consequence of damage. Intensity fluctuations at high dose rates may arise from the impulsive release of gaseous damage products. Accounting for these effects, data collection at the highest dose rates increases crystal radiation lifetimes near 300 K (but not at 100 K) by a factor of ∼1.5-2 compared with those observed at conventional dose rates. Improved quantification and modeling of the complex spatio-temporal evolution of protein microcrystal diffraction in intense microbeams will enable more efficient data collection, and will be essential in improving the accuracy of structure factors and structural models.

Keywords: X-ray crystallography; intense X-ray microbeams; microcrystallography; protein crystallography; protein structure; radiation damage; serial crystallography; structural biology; structure determination.

Figure 1

Representative semi-log plot of the integrated intensity in diffraction peaks versus dose at several dose rates, acquired from a single, fixed-orientation lysozyme crystal at 300 K; Supplementary Fig. S5 shows 260 K data. Solid lines are single-parameter fits at the highest and lowest dose rates based on the model described here; dashed lines indicate the initial exponential trend. The intersection of the horizontal dashed black line with each dose curve determines the half-dose D _1/2. Doses and dose rates in all figures are averages within the area of the FWHM of the Gaussian beam.

Figure 2

Top: representative integrated intensity versus dose data for two thaumatin crystals at 260 K for a dose rate of 0.09 MGy s⁻¹. Each curve was recorded from one sample position. Sample 1 has an intensity variation with dose as in Fig. 1 ▸, while sample 2 has an initial plateau in intensity. Bottom: the intensity plateau for sample 2 results from initial growth with increasing dose of a subset of Bragg peaks that dominate the integrated intensity. Supplementary Fig. S6 shows similar data acquired at a dose rate of 36 MGy s⁻¹.

Figure 3

Half-dose versus dose rate for tetragonal lysozyme at 100, 260 and 300 K. Similar plots are obtained for thaumatin, and half-doses are summarized in Supplementary Table S1. Each dose-rate point represents an average of half-doses determined from between five and 35 dose curves obtained from different positions on each sample. The different symbols at each temperature indicate data from different samples. The error bar on each point represents the corresponding standard deviation.

Figure 4

Top: diffracted flux from a crystal, proportional to the integrated intensity in Bragg peaks, versus dose calculated for a Gaussian beam and for a beam with a top-hat (rectangular) profile of width equal to the Gaussian FWHM, for a fixed crystal orientation during irradiation. The local decay of diffraction with dose is assumed to be exponential with a half-dose D _1/2,local equal to that measured with a top-hat profile beam. Bottom: diffraction-weighted dose DWD* (Zeldin, Brockhauser et al., 2013 ▸) and DWD (as revised here) versus normalized dose for a Gaussian beam and a fixed crystal.

Figure 5

Calculated diffracted flux per unit beam area (top) and per unit beam radius versus radius (bottom) for equally spaced nominal doses for a Gaussian beam and a locally exponential decay of diffraction with dose.