Fig. 1. Double-exchange events can be observed by smFRET.
a, Schematic of the assay. Nucleosomes (113N2.AF488) labelled with AF488 (blue) on the short 2-bp overhang are surface immobilized on a PEGylated microscope slide. SWR1, ATP and AF555–Htz1–H2B dimers (green) are flowed in to start the exchange reaction. Histone exchange is detected as a FRET increase between AF488 and AF555. b, Intensity trajectory (top) and corresponding FRET trajectory (bottom) for a single nucleosome showing a stepwise gain in FRET signal following each dimer exchange. c, Idealized FRET histogram of the first-exchange event shows two approximately equal populations of approximately 0.4 and approximately 0.6 FRET corresponding to either dye-distal or dye-proximal exchange. d, Dwell time distribution between the first and second exchanges yields a second-exchange time τ2 = 246 ± 25 s. Reported errors are the error of the fit.