Fig. 1.
(A) Crystal structure of the cytosolic part of syntaxin in complex with munc-18. Munc-18 and the transmembrane region are not shown. Yellow, Habc domain and linker region; red, SNARE motif (H3 domain). Black spheres indicate residues (numbers) substituted by cysteines for dye labeling. (B–E) SmMFD analysis of burst-averaged observables (number of bursts increases from white to black) for different FRET experiments (Upper) with SNARE proteins (sketched below). (B) The sketched new combination of different observables and their specific advantages in precision and accuracy allow to measure absolute interdye distances, _R_DA. In particular, the two main sources of errors in distance calculations, individual local quenching effects (Upper) and restricted mobility of the reporter dyes (Lower), can be excluded by SmMFD. First, in 
plots, local quenching of D and A, respectively (green and red arrows), is distinguished from distance-dependent FRET effects (red sigmoidal curve: Eq. 1 with 
, kFD = 1.6 × 108s–1, R_0_r = 59.1 Å). Second, by connecting two-dimensional plots of 
vs. the donor anisotropy (_r_D) allows one to analyze the dye mobilities (overlaid red curve) not only for the ensemble but also for all substates by computing the average rotation correlation times of the donor (ρD) by using the Perrin equation, 
, assuming an anisotropy value at time 0 for rhodamine dyes of _r_0 = 0.375). (C–E) Following ref. , the net fluorescence signal is obtained by correcting the measured green and red signal for the background, BG = 7.0 kHz, and BR = 3.5 kHz, spectral crosstalk, α = 0.035, and ratio of green and red detection efficiencies, _g_R/_g_G = 1.25. _B_R also includes direct acceptor excitation, DE = 1.0 kHz. Histograms obtained for Sx 91/225 in complex with munc-18 (C), alone (D), and as part of the ternary SNARE complex (E). Also shown are projections yielding one-dimensional distributions of 
(black, Upper), _R_DA (magenta, Right Upper), and _r_D (dark blue, Right Lower). The measured distance distributions (C–E) are broadened by shot noise and also for certain label positions by structural dynamics. This result is shown in the projected _R_DA distributions giving the calculated shot noise described by a Gaussian distribution with SD σ(_R_DA) (black lines, equation 5 of ref. 17) and the fit to two Gaussian distributions (yellow lines, D). Molecules containing only the donor dye exhibit apparent distances larger than 85 Å and were excluded from further analysis. For all different syntaxin mutants, environments, and conformations, significant dye interactions can be excluded: (i) no detectable specific local quenching (Upper); and (ii) the average rotation correlation times of the donors, ρD, are small for all substates (Lower), which proves the value of the FRET orientation factor, κ2, of 2/3.