Spectroscopic measurement series typically have to cover multidimensional parameter spaces, and a rapid overview of the collected data is paramount in making maximum use of precious beamtime.

While synchrotron users are often proficient in interpreting solution spectroscopic data, the particular artefacts observed in the spectra of samples in crystalline form can be easily misinterpreted. The combination of in situ spectroscopies with X-ray-induced photophysics allows the characterization of transient states accessible by electron transfer (Schlichting et al., 2000 Hough et al., 2008 Adam et al., 2009 Hersleth & Andersson, 2011 ), monitoring of the oxidation states of metal centres (Beitlich et al., 2007 Ellis et al., 2008 He et al., 2012 Merlino et al., 2013 ), quantification of radiation damage (Meents et al., 2007 Rajendran et al., 2011 McGeehan et al., 2007 ) and the general understanding of its mechanisms (Carpentier et al., 2010 Sutton et al., 2013 ).

A particularly useful application of complementary techniques is the assessment of X-ray-induced photophysics in the sample, also known as radiation damage. Spectroscopies successfully applied to protein crystals in situ include UV–visible absorption, fluorescence, resonance Raman and nonresonance Raman as well as X-ray absorption spectroscopy. Examples include instruments at the ESRF (Carpentier et al., 2007 Royant et al., 2007 Davies et al., 2009 ), APS (Pearson et al., 2007 ), NSLS (Stoner-Ma et al., 2011 Orville et al., 2011 ), DLS (Allan et al., 2013 ), SPring-8 (Sakai et al., 2002 Shimizu et al., 2013 ) and SLS (Owen et al., 2009 Pompidor et al., 2013 ). Since its first introduction in the 1970s (Rossi & Bernhard, 1970 ) it has come a long way, and today is implemented in several experimental endstations of macromolecular crystallography beamlines at storage rings around the globe, allowing a larger community of structural biologists to benefit from the opportunities provided by this complementary method. In situ micro-spectrophotometry is a highly effective way to obtain such complementary information about the chemical identity of cofactors, the electronic state of metal centers or about specific radiation-induced chemistry. This is especially true in the fields of enzyme kinetics and protein function, ligand-binding modes or redox states of metal centres. The structural information provided by electron-density maps obtained from diffraction experiments often does not provide sufficient insight to answer the point of interest conclusively. Complementary biophysical techniques are a powerful tool to complete and enhance structural information obtained by macromolecular crystallography (MX).

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