Abstract
The local Fourier-space relation between diffracted intensity I, diffraction wavevector q and dose D, , is key to probing and understanding radiation damage by X-rays and energetic particles in both diffraction and imaging experiments. The models used in protein crystallography for the last 50 years provide good fits to experimental I(q) versus nominal dose data, but have unclear physical significance. More recently, a fit to diffraction and imaging experiments suggested that the maximum tolerable dose varies as q -1 or linearly with resolution. Here, it is shown that crystallographic data have been strongly perturbed by the effects of spatially nonuniform crystal irradiation and diffraction during data collection. Reanalysis shows that these data are consistent with a purely exponential local dose dependence, = I 0(q)exp[-D/D e(q)], where D e(q) ∝ q α with α ≃ 1.7. A physics-based model for radiation damage, in which damage events occurring at random locations within a sample each cause energy deposition and blurring of the electron density within a small volume, predicts this exponential variation with dose for all q values and a decay exponent α ≃ 2 in two and three dimensions, roughly consistent with both diffraction and imaging experiments over more than two orders of magnitude in resolution. The B-factor model used to account for radiation damage in crystallographic scaling programs is consistent with α = 2, but may not accurately capture the dose dependencies of structure factors under typical nonuniform illumination conditions. The strong q dependence of radiation-induced diffraction decays implies that the previously proposed 20-30 MGy dose limit for protein crystallography should be replaced by a resolution-dependent dose limit that, for atomic resolution data sets, will be much smaller. The results suggest that the physics underlying basic experimental trends in radiation damage at T ≃ 100 K is straightforward and universal. Deviations of the local I(q, D) from strictly exponential behavior may provide mechanistic insights, especially into the radiation-damage processes responsible for the greatly increased radiation sensitivity observed at T ≃ 300 K.
Highlights
Radiation damage is a key issue in all diffraction and imaging methods that illuminate biological samples with energetic particles such as X-ray photons, electrons, neutrons and positrons
By assuming a purely exponential local dependence of diffracted intensity on dose of the form I~ðq; DÞ = I0(q) exp[ÀD/De(q)] with De(q) = K/q, and accounting for the nonuniform pattern of crystal irradiation during data collection, we obtain good fits to experimental I(q) versus nominal dose/fluence relations measured for several protein crystals at
Deviations of the calculated dose/fluence dependence from the data may arise because the actual crystal shapes and initial crystal orientations deviate from those assumed, and because of issues in data collection and processing that cause measured intensities to deviate from the actual dose-dependent structure factors (Warkentin et al, 2017)
Summary
Radiation damage is a key issue in all diffraction and imaging methods that illuminate biological samples with energetic particles such as X-ray photons, electrons, neutrons and positrons. Accumulation of bond-scale damage causes degradation of sample order on larger and larger length scales (Warkentin et al, 2013). Damage manifests as a decrease in the diffracted intensity at large angles 2 or large diffraction wavevectors q that progresses to smaller and smaller q. In addition to these ‘global’ effects of radiation damage, metal sites, disulfide bonds and other structures within the sample may be sensitive to damage, giving rise to ‘site-specific’ damage (Ravelli & McSweeney, 2000; Weik et al, 2000; Burmeister, 2000; Banumathi et al, 2004)
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