Fig. 2
From: The architecture of EGFR’s basal complexes reveals autoinhibition mechanisms in dimers and oligomers
FLImP measurement of pairwise DIII-binding Affibody separations. a Cartoon of FLImP histograms. Left: A dimer (receptors, blue; label, red) gives rise to one separation, the empirical posterior of which has a confidence interval (CI) that depends on the combined localisation errors of the two molecules28. CI size determines resolution. CIs less than the required resolution are retained according to signal-to-noise without bias12,28,29,30,36, generating a FLImP distribution (grey) that is fitted by the sum of a number of Rician peaks12. If oligomers are present and internally probed (middle) and/or the distribution of species is inhomogeneous (right), the histogram contains multiple components12. b FLImP distribution (grey) of DIII–DIII separations between CF640R-Affibody molecules bound to wtEGFR on CHO cells, compiled from 68 FLImP measurements (CI ≤ 7 nm), decomposed into a sum of five components (coloured traces). The inset shows positions and error estimates. Additional statistics in Supplementary Fig. 1. The 4 nM concentration of CF640R-Affibody used labels ~20% of receptors (Supplementary Fig. 2) ensuring single particle detection29. FLImP is stochastic, thus independent of the CF640R-Affibody/receptor ratio if sufficient data are collected, and uses fixed cells to avoid relative receptor movements during measurements. However, systematic studies failed to find significant artifacts12,36. c Molecular-normalised fraction of receptors in oligomer species on wtEGFR-expressing CHO cells treated with 100 nM Alexa 488-Affibody, determined by pbICS38. Results are the mean of seven replicates. Error bars show the SD. For more details see Supplementary Fig. 3. d Cartoon showing expected FLImP distributions for a cyclic tetramer labelled with a 1:1 probe binder (like the Affibody) (left), a polymer chain labelled with a 1:1 probe binder (middle), and a polymer showing a 2N:2 labelling stoichiometry (like EGF)12 (N = receptor number) (right). e FLImP distribution (grey) and peak decomposition of DIII–DIII separations reported by CF640R-Affibody molecules bound to I942E-EGFR on the surface of CHO cells, compiled from 36 FLImP measurements (CI ≤ 7 nm), decomposed into a sum of four peak components (coloured traces). Positions and error estimates are shown in the inset. f As e using 4 nM CF640R-EGF as a probe, compiled from 31 FLImP measurements (CI ≤ 7 nm)
