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A Raman micro­spectro­photometer is described that allows the spectroscopic investigation of protein crystals under exactly the same conditions as those used for X-ray data collection. The concept is based on the integration of the Raman excitation/collection optics into a micro­spectro­photometer built around a single-axis diffractometer and a cooling device. It is shown that Raman spectra of outstanding quality can be recorded from crystallized macromolecules under non-resonant conditions. It is proposed that equipment developed in the context of macromolecular cryo­crystallography, such as commonly used cryoloops, can be advantageously used to improve the quality of Raman spectra. In a few examples, it is shown that Raman micro­spectro­photometry provides crucial complementary information to X-ray crystallography, e.g. identifying the chemical nature of unknown features discovered in electron-density maps, or following ligand-binding kinetics in biological crystals. The feasibility of `online' Raman measurements performed directly on the ESRF macromolecular crystallography beamlines has been investigated and constitutes a promising perspective for the routine implementation of combined spectroscopic and crystallographic methods. In crystallo Raman spectroscopy efficiently complements absorption/fluorescence micro­spectro­photometry for the study of biological crystals and opens up new avenues for difficult structural projects with mechanistic perspectives in the field of protein crystallography.

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