Advances in spectroscopic methods for biological crystals. 2. Raman spectroscopy

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 singleaxis 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.