CTD dimerization of Hsp90 is necessary for its function. To study the effect of 5b exposure on the dissociation of Hsp90 dimers in a cell-free assay, we used Hsp90α CTD protein after incubation with amine-reactive crosslinker BS 3 , as previously described. A dose-dependent reduction of Hsp90α CTD dimers along with an increase in the high order oligomeric species was noticed upon incubation with 5b (Figure ). Next, we performed small-angle X-ray scattering (SAXS) with the Hsp90α CTD protein, which was coupled to a size exclusion chromatography column (SEC-SAXS) at the ESRF beamline BM29 in Grenoble. In the absence of 5b, a clear dimeric profile of the Hsp90α CTD protein was visible on the chromatogram, with an additionally minor tetrameric species (SI Figure ). We used the program CHROMIXS to merge the frames containing the dimer from this SEC-SAXS profile.