Single-Molecule FRET Measurements Protocol

Single-molecule FRET measurements were performed at room temperature using a home-built confocal microscope (as previously described 35 (link)). Briefly, the microscope operated with 20 kHz alternating-laser excitation between a 532-nm (Samba, Cobolt, operated at 240 μW) and a 638-nm laser (Cube, Coherent, operated at 60 μW), coupled to a 60×, 1.35 numerical aperture (NA), UPLSAPO 60XO objective (Olympus). Emitted photons were spectrally filtered and detected by two avalanche photo diodes (SPCM-AQRH-14, Perkin Elmer). The alternating laser excitation allows filtering for correctly labelled species bearing an active donor and an active acceptor (36 (link)). After filtering each fluorescent burst for the correct labelling stoichiometry, we calculated the apparent FRET efficiency E* for each burst as E* = DA/(DD + DA), where DA is the number of photons in the red detection channel after green excitation and DD the number of photons in green detection channel after green excitation.