Although anomalous scattering, site-specific radiation damage and AAS in the substructure are different phenomena with different physical origins, they have the common property of inducing intensity differences between symmetry-related reflections in a diffraction experiment. Provided that these effects are included in a parametrized model of the substructure, they can become a source of phase information. With the current practice of recording diffraction data using the single-axis rotation method, it is almost always the case that data sets with a certain degree of redundancy (multiplicity) are collected. Redundancy is often achieved before completeness, i.e. several symmetry-equivalent observations of certain reflections are recorded while for other reflections no observations have yet been measured. Thus, redundancy is usually a byproduct of striving to collect a complete data set. In this sense, the additional phase information that may be contained in the intensity differences between symmetry-related reflections essentially comes ‘for free’. Since overall radiation damage is in many cases the main limiting factor in the amount of data that can be collected from a single sample for the purpose of experimental phasing, it is of the utmost importance to be able to derive the maximum amount of phase information from this limited amount of data. A current limitation in the implementation of these methods is the approximate treatment of correlated sources of non-isomorphism.
Although in many cases the standard anomalous signal generated through Bijvoet differences is likely to be the main source of phase information, this can be supplemented by exploiting the symmetry-breaking effects in unmerged data as described above. In particular cases, such as that of the brominated RNA fragment described earlier, site-specific and overall radiation damage evolve on significantly different timescales. The former can then become a very significant source of phase information to complement the anomalous phasing signal. However, such favourable cases are rather atypical. In many ‘real-life’ situations overall radiation damage unfolds at a rate that is not significantly different in comparison to the evolution of site-specific radiation damage or in comparison to the total time that is required to record a complete data set. In such cases the quality of the phases will ultimately be limited by the effects of overall radiation damage, although the proper modelling and exploitation of site-specific radiation damage can still yield a noticeable improvement of phases, as was for instance the case in the structure determination of the PP2A phosphatase activator Ypa2 (Leulliot et al.
; Schiltz & Bricogne, 2007
The general question then arises of how to design a data-collection strategy that enables the optimal exploitation of the various possible sources of phase information when the lifetime of the crystal is limited. Crystals can be intentionally misaligned in order to maximize the AAS-induced inequivalence between symmetry-related reflections. However, since the standard anomalous signal is the most important source of phase information, the reduction of systematic errors in Bijvoet intensity differences is of prime importance. In crystals belonging to high-symmetry space groups, Bijvoet pairs are usually recorded in close temporal proximity, even if the crystal is not specially aligned. In such cases there will always be some symmetries that are broken by AAS and these effects can then be exploited for additional phase information. For crystals of lower symmetry the deliberate alignment of a symmetry axis along the spindle allows the simultaneous recording of Bijvoet pairs. However, if the spindle is oriented horizontally (i.e. along the direction of linear polarization of the synchrotron beam) such a geometry will partly or fully neutralize the symmetry-breaking effects of AAS. A more ideal geometry, which would minimize systematic errors in Bijvoet intensity differences by aligning a symmetry axis with respect to the spindle axis, while at the same time maximizing the symmetry-breaking effects of AAS by misaligning the symmetry axis with respect to the direction of X-ray polarization, can be achieved with multi-axis goniometers, where the scan axis is not constrained to be aligned with the X-ray polarization direction. Thus, with the future use of goniometers with a vertical scan axis designed for the purposes of gaining mechanical stability in the handling of microcrystals, the effects of AAS will become truly ubiquitous in all data sets collected at an absorption edge of a covalently bonded anomalous scatterer and their proper treatment in experimental phasing will be imperative if a major waste of phase information is to be avoided.