(a) Illustration of the single-molecule FRET experiment using an objective-type TIRF microscope (bottom): cross section through the objective, and flow chamber (both gray) and the dichroic mirror (black) separating the laser excitation (green) from the collected fluorescence (yellow). The zoom view (top) shows the fluorescently labeled Hsp90 (FRET donor, orange; acceptor red), which is immobilized on a PEG-passivated (dark blue) coverslip (light blue) using biotin-neutravidin coupling (black and gray). (b) Example time traces obtained from individual Hsp90 molecules for the point mutant A577I (top), in the presence of 3.5 µM cochaperone Aha1 (center), or under macro-molecular crowding by 20wt% Ficoll400 (bottom). Depicted are the FRET efficiency E (black), the fluorescence of the FRET donor (green) and acceptor (red) and the directly excited acceptor (gray). White and colored overlays denote low- and high-FRET dwells, respectively, as obtained using a hidden Markov model and the Viterbi algorithm. (c) FRET histograms compiled from many single-molecule trajectories as indicated, and normalized to unity (wt: wild type). Reference data (black) were measured under the specific conditions of each of the three experiment series (see also Materials and methods). Example traces of the reference data are provided in Figure 2—figure supplement 1. All fits and fit coefficients for 2 c are shown in Figure 2—figure supplement 2. Experimental conditions for A577I or wt Hsp90: 2 mM ATP in 40 mM Hepes, 150 mM KCl, 10 mM MgCl2, pH7.5. Conditions with and without 3.5 µM Aha1: wt Hsp90, 2 mM ATP in 40 mM Hepes, 20 mM KCl, 5 mM MgCl2, pH 7.5. Conditions with and without crowding by 20wt% Ficoll400: wt Hsp90 in 40 mM Hepes, 150 mM KCl, 10 mM MgCl2, pH7.5. The data were recorded in 5 to 13 videos per dataset, on one or more days. The number of individual molecules included per histogram are: A577I, 154; wt, 163; +Aha1, 366; -Aha1, 231; +crowding, 50; -crowding, 81. All smFRET traces are available from Figure 2—source data 1.