Research Article: X-ray and UV radiation-damage-induced phasing using synchrotron serial crystallography

Date Published: April 01, 2018

Publisher: International Union of Crystallography

Author(s): Nicolas Foos, Carolin Seuring, Robin Schubert, Anja Burkhardt, Olof Svensson, Alke Meents, Henry N. Chapman, Max H. Nanao.


Multi-crystal serial crystallography data can be used for UV and X-ray radiation-damage-induced phasing.

Partial Text

Radiation induces many changes in macromolecular crystals. Amongst these is a reduction in occupancy or the movement of atoms, which is referred to as specific radiation damage. Specific radiation damage can be induced by X-ray or UV light and affects metals, Sγ atoms in disulfides, thiol linkages and terminal O atoms in carboxylates (with the latter only being induced by X-rays; Ravelli & McSweeney, 2000 ▸; Burmeister, 2000 ▸; Weik et al., 2000 ▸; Pattison & Davies, 2006 ▸). Specific radiation damage can be of major concern to practitioners of macromolecular crystallography (MX), but in some cases such damage can be used to determine phases experimentally (Ravelli et al., 2003 ▸, 2005 ▸; Zwart et al., 2004 ▸; Banumathi et al., 2004 ▸; Weiss et al., 2004 ▸; Schiltz et al., 2004 ▸; Ramagopal et al., 2005 ▸; de Sanctis & Nanao, 2012 ▸; de Sanctis et al., 2016 ▸). This technique is called radiation-damage-induced phasing (RIP) and, by analogy to single isomorphous replace­ment (SIR), two data sets are used to calculate differences in structure factors (between damaged and less damaged states). However, unlike in SIR, no soaking of heavy atoms is required. If the decrease in occupancy at specific sites is large enough and global radiation damage has been minimized, the positions of radiation damage can be determined. UV RIP generally has the advantage of inducing less general global radiation damage compared with X-ray RIP (Nanao & Ravelli, 2006 ▸; de Sanctis et al., 2016 ▸). When performed on a single crystal or indeed at the same position of single crystals, RIP has the advantage of relatively high isomorphism between the damaged and undamaged data sets. This is a key difference between RIP and traditional isomorphous methods, in which the experiment is performed on different crystals and the introduction of a heavy atom frequently introduces non-isomorphism. Depending on the ratio of specific to global damage, the number of sites and their susceptibility, a wide range of relative changes to intensities can be expected. Initial estimates of the maximal signal based on Crick & Magdoff (1956 ▸) suggested that even modest reductions to occupancies of 26% for six disulfide S atoms could lead to changes in intensity of 10% at 2θ = 0 (Crick & Magdoff, 1956 ▸; Ravelli et al., 2003 ▸). In practice, a wide range of R values between damaged and undamaged data sets have been observed: up to 14% overall for trypsin despite low (∼4%) internal R values (Nanao et al., 2005 ▸). This differentiates RIP from the other dominant phasing method based on endogenous chemical groups: long-wavelength sulfur SAD. Thus, the potentially high signal and the lack of a requirement for chemical modification of crystals provides a potentially useful alternative method to traditional isomorphous and anomalous methods. However, one key limitation of X-ray and UV RIP approaches is that a minimum of two complete data sets must normally be collected. Two solutions to this limitation are to collect one large data set and subdivide it into two sub-data sets in a ‘segmented RIP’ analysis (de Sanctis & Nanao, 2012 ▸) or to model specific damage as a function of dose, as in SHARP (Schiltz et al., 2004 ▸; Schiltz & Bricogne, 2008 ▸, 2010 ▸). In segmented RIP, one collects a large high total dose data set, and the first images collected are treated as a low-damage data set and the last images are treated as a damaged data set. Finally, in cases of large crystals, multiple positions can be collected from a single crystal, allowing the measurement of one complete low-damage data set prior to UV/X-ray exposure. However, the utility of this approach is limited by the trend towards smaller crystals, as well as by intra-crystal non-isomorphism. In UV RIP experiments, the amount of damage depends on the UV source, on the composition of the unit cell and on the crystal volume. In particular, the limited light-penetration depth in macromolecular crystals is a significant challenge to the homogenous illumination of larger crystals. Thus, using small crystals has significant advantages if complete data sets can be collected. While penetration depth is not an issue for X-ray damage, improvements to phasing can be expected if high-multiplicity data sets can be collected. To this end, we have employed recent developments in synchrotron serial crystallography (SSX) to greatly increase the recorded signal at a given dose by combining data from multiple crystals (Diederichs & Wang, 2017 ▸). A major challenge in implementing SSX-RIP is to efficiently deal with non-isomorphism between crystals. Simulated diffraction patterns for free-electron laser serial femtosecond crystallography (SFX), where there is no rotation during exposure, have indicated that such an approach is possible, but it has not yet been demonstrated experimentally (Galli, Son, White et al., 2015 ▸; Galli, Son, Barends et al., 2015 ▸). Here, we show for the first time that SSX can be used to successfully phase macromolecular crystals of thaumatin and insulin de novo by X-ray RIP and UV RIP, and explore the relationship between dose, multiplicity and RIP signal.

RIP offers a complementary method to traditional anomalous and isomorphous methods for the experimental determination of phases. Although RIP can also be used in combination with anomalous and isomorphous methods, it is a useful method on its own, particularly when heavy-atom derivatization or seleno­methionine substitution is difficult. Recent advances in multiple-crystal techniques have made it practical to determine high-resolution structures from X-ray data acquired from a large number of crystals. Here, we show that a serial approach yields sufficient signal to determine phases de novo by both X-ray RIP and UV RIP for these two test systems. In this study, we have assembled low-dose and high-dose data sets independently; however, we are also exploring the possibility of improving RIP signal by optimizing both data sets simultaneously. In this way, the isomorphous signal could be improved depending on which sub-data sets are selected. Because of the relatively high symmetry of thaumatin and insulin, we have simply used the strongest sub-data set as a reference for indexing other sub-data sets during processing. However, in some cases an individual sub-data set might not contain enough reflections for this purpose. In these cases, alternate methods for indexing (and resolving indexing ambiguity) might become necessary, for example using the method developed by Brehm & Diederichs (2014 ▸). For very incomplete sub-data sets, scaling becomes impossible owing to a lack of common reflections, unless a reference data set is available.




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