Chapter 16 Raman-Assisted X-Ray Crystallography for the Analysis of Biomolecules Dominique Bourgeois, Gergely Katona, Eve de Rosny, and Philippe Carpentier Summary In this chapter, we describe Raman microspectrophotometry applied to crystals of biomolecules. Raman spectra collected in crystallo provide structural information highly complementary to X-ray diffraction, relate the crystalline state to the solution state, and allow the identification of ligand-bound or interme- diate states of macromolecules. Nonresonant Raman spectroscopy is particularly suitable to the study of macromolecular crystals, and therefore applies to a wide range of noncolored crystalline proteins. Practi- cal issues related to the investigation of crystals by Raman microspectrophotometry are reviewed, and the current limitations are highlighted. Key words: In crystallo Raman spectroscopy , Macromolecules, Microspectrophotometers, Crystallography , Complementary methods, Nonresonant Raman spectroscopy Applying complementary techniques that probe different proper- ties of the same biomolecule helps understanding the relationship between structure, dynamics, and function. Optical spectroscopy, encompassing the ultraviolet (UV) to infrared (IR) range, con- stitutes a particularly powerful tool to be used jointly with X-ray crystallography, the central technique to decipher the three-di- mensional (3D) structure of biological macromolecules. Micro- spectrophotometers have been developed to investigate protein crystals in experimental conditions identical to those typically 1. Introduction James Weifu Lee and Robert S. Foote (eds.), Micro and Nano Technologies in Bioanalysis, Methods in Molecular Biology, vol. 544 DOI 10.1007/978-1-59745-483-4_16, © Humana Press, a part of Springer Science + Business Media, LLC 2009 253