Towards Structural Biology with Single Molecules

X‐ray diffraction crystallography and nuclear magnetic resonance can provide high‐resolution structural information about macromolecular systems. However, these methods determine the state of a large ensemble of molecules, essentially providing averages of atomic positions. In contrast, single molecule fluorescence experiments enable investigations of macromolecular conformation and folding of individual molecules by the introduction of fluorescent dyes at specific sites in the macromolecule. Multiple such experiments can be performed with different labeling site combinations in order to map complex conformational changes or interactions between multiple molecules. When combined with known structures of the macromolecules to which the fluorophores are attached, a three‐dimensional model of the macromolecular complex can be determined using computational approaches.