Single-molecule fluorescence resonance energy transfer (smFRET) is an important tool in structural biology, used to report the proximity of two probes whose fluorescence readout is modulated by distance. Ratzke et al. now expand the use of smFRET to four colors to study conformational changes in the Hsp90 protein-complex chaperone from yeast. They used fluorescent Atto dyes to label the two Hsp90 monomers, cochaperone p23 and ATP in order to detect p23 docking, changes in Hsp90 configuration, ATP binding and the presence of all three components. The researchers found that p23 binds to Hsp90 in the presence of ATP and that ATP hydrolysis causes a rearrangement of the binding partners in a directional manner.