Phasing in the presence of radiation damage

In the accurate estimation of small signals, redundancy of observations is often seen as an essential tool for the experimenter. This is particularly true during macromolecular structure determination by single-wavelength anomalous dispersion ( SAD), where the exploitable signal can be less than a few percent. At the most intense undulator synchrotron beamlines, the effect of radiation damage can be such that all usable signal is obscured. Here the magnitude of this effect in experiments performed at the Se K-edge is quantified. Six successive data sets were collected on the same crystal, interspersed with two exposures to the X-ray beam during which data were not collected. It is shown that the very first data set has excellent phasing statistics, whereas these statistics degrade for the later data sets. Merging several data sets into one, highly redundant, data set only gave moderate improvements as a result of the presence of radiation damage. Part of the damage could be corrected for using a linear interpolation scheme. Interpolation of the data to a low-dose as well as to a high-dose data set allowed us to combine the SAD method with the radiation-damage induced phasing ( RIP) technique, which further improved the experimental phases, especially after density modification. Some recommendations are given on how to mitigate the effect of radiation damage during structure determination.