Nat. Struct. Mol. Biol. 26, 802–807 (2019).

Measuring small conformational changes within proteins, that occur at millisecond timescales, is extremely challenging. However, the problem of resolving such motions can often be reduced to resolving unique spatial orientations of a single key structural element, such as an α-helix. Lewis et al. attach a fluorophore to the protein-element of interest and track changes in its orientation using a polarization microscope. Collecting fluorescence intensities at a high rate of 50 frames per second allowed them to quantify real-time motions in a single protein. They analyzed the transitions between the three conformational states of the RCK domain of the prokaryotic Ca2+-dependent K+ channel MthK, and were able to observe states that differed by 3–8 Å. The success of the method depends on obtaining a signal-to-noise ratio that works for the desired resolution, and on choosing a structural element that is representative of the conformational changes under observation.