title: Challenges and solutions for the analysis of in situ, in crystallo micro-spectrophotometric data.
abstract: Combining macromolecular crystallography with in crystallo micro-spectrophotometry yields valuable complementary information on the sample, including the redox states of metal cofactors, the identification of bound ligands and the onset and strength of undesired photochemistry, also known as radiation damage. However, the analysis and processing of the resulting data differs significantly from the approaches used for solution spectrophotometric data. The varying size and shape of the sample, together with the suboptimal sample environment, the lack of proper reference signals and the general influence of the X-ray beam on the sample have to be considered and carefully corrected for. In the present article, how to characterize and treat these sample-dependent artefacts in a reproducible manner is discussed and the SLS-APE in situ, in crystallo optical spectroscopy data-analysis toolbox is demonstrated.
authors.affiliation: Swiss Light Source, Paul Scherrer Institute, CH-5232 Villigen PSI, Switzerland.
authors.forename: Florian S N
authors.initials: FS
authors.lastname: Dworkowski
authors.affiliation: School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, England.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Hough
authors.affiliation: European Molecular Biology Laboratory Hamburg, c/o DESY, Notkestrasse 85, D-22603 Hamburg, Germany.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Pompidor
authors.affiliation: Photon Sciences, Brookhaven National Laboratory, Mail Stop 745, Upton, NY 11973, USA.
authors.forename: Martin R
authors.initials: MR
authors.lastname: Fuchs
mesh_terms: D001331:Automation; D003461:Crystallography / Q000379:methods*; D005453:Fluorescence; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D013059:Spectrum Analysis, Raman / Q000379:methods*
keywords: Raman spectroscopy; UV–visible absorption; background correction; data analysis; micro-spectrophotometer; single-crystal spectroscopy; software toolbox