Absorbed dose calculations for macromolecular crystals: improvements to RADDOSE

Abstract

Radiation damage is an unwelcome and unavoidable aspect of macromolecular crystallography. In order to quantify the extent of X-ray-induced changes, knowledge of the dose (absorbed energy per unit mass) is necessary since it is the obvious metric against which to plot variables such as diffraction intensity loss and B factors. Significant improvements to the program RADDOSE for accurately calculating the dose absorbed by macromolecular crystals are presented here. Specifically, the probability of energy loss through the escape of fluorescent photons from de-excitation of an atom following photoelectric absorption is now included. For lighter elements, both the probability of fluorescence and of its subsequent escape from the crystal are negligible, but for heavier atoms the chance of fluorescence becomes significant (e.g. 30% as opposed to Auger electron decay from a K-shell excited iron atom), and this has the effect of reducing the absorbed dose. The effects of this phenomenon on dose calculations are presented for examples of crystals of an iron-containing protein, 2-selenomethionine proteins, a uranium derivatised protein, and for a nucleic acid sample. For instance, the inclusion of fluorescent escape results in up to a 27% decrease in the calculated absorbed dose for a typical selenomethionine protein crystal irradiated at the selenium K-edge.

Determination of X-ray flux using silicon pin diodes

“…This calculation makes several assumptions, not least concerning the beam size and shape, but does provide a rough (correct within a factor of two for most samples not containing heavy atoms) indication of the time that a macromolecular crystal exposed to such a beam will last before absorbing the experimental dose limit of 30 MGy (reduction of initial diffraction intensity, I 0 to 0.7I 0 ), after which diffraction data will have questionable value (Owen et al, 2006). For accurate dose determination, RADDOSE should be used with appropriate input values for the beam size, shape and profile, and crystal parameters (Paithankar et al, 2009;Murray et al, 2004).…”

“…The dose absorbed by a crystal can be calculated from the physics of the interaction between X-rays and atoms, but a prerequisite to this calculation is knowledge of the size, shape and intensity (flux: photons s À1 ) of the incident X-ray beam (Murray et al, 2004;Paithankar et al, 2009). These parameters are not yet routinely measured at all synchrotron MX beamlines, and this paper will focus on how accurate determination of X-ray photon flux can be achieved.…”

Predicting the X-ray lifetime of protein crystals

“…Two intuitive metrics are the average dose for the whole crystal volume (AD-WC); i.e., the total absorbed energy divided by the mass of the whole crystal, and the maximum dose: the highest dose reached at any point in the crystal volume. Maximum dose, assuming a well-aligned beam and rotation axis, is the metric output by previous versions of RADDOSE (13)(14)(15) when using the Gaussian beam profile (GAUSS) keyword to define the beam profile, a worst-case estimate for the dose.…”

Diffraction data analysis in the presence of radiation damage

“…A good example of such analysis is the program RADDOSE, which calculates the X-ray absorption depending on the crystallization conditions, the particular composition of the protein and the X-ray wavelength (Murray et al, 2005;Paithankar et al, 2009). The results of this and similar analyses are used as one of the intermediate steps in the process of planning the experiment.…”