(a) iSCAT/TIRF microscopy image of unlabeled wildtype microtubules (WT, magenta) growing from GMPCPP-seeds at 12 µM wildtype human tubulin in the presence of 20 nM human mGFP-EB3 (green). (b) Kymographs showing mGFP-EB3 (green) tracking the ends of growing wildtype microtubules, conditions as in (a). (c) Mean mGFP-EB3 intensity profiles at the ends of human wildtype microtubules (WT MT) growing at 12 µM wildtype tubulin in the presence of varying mGFP-EB3 concentrations (as indicated). Number of microtubules (and frames) averaged for each mGFP-EB3 concentration: 2 nM – 64 (7360), 5 nM – 87 (9222), 10 nM – 93 (8742), 25 nM – 89 (8900), 50 nM – 56 (5600), 100 nM – 58 (4756), 200 nM – 56 (4088), 500 nM – 46 (4140). Error bars are SD. (d) Quantification of mGFP-EB3 binding affinity to microtubule ends growing at 12 µM wildtype tubulin (WT MT). Black symbols depict the averaged maximal mGFP-EB3 intensities at growing plus ends at each condition, error bars represent SD. Experimental data as in (c). A hyperbolic fit (cyan) was used to calculate the Kd. (e) mGFP-EB3 does not affect significantly the growth speed of human wildtype microtubules (WT MT). Experimental data as in (c). (f) iSCAT/TIRF microscopy images of 20 nM mGFP-EB3 (green) binding to unlabeled E254A mutant microtubules (magenta) attached to a Kin1rigor surface. (g) Magnified images and (h) kymographs of stable unlabeled E254A microtubules (magenta) decorated with 20 nM mGFP-EB3 (green), conditions as in (d). (i) Quantification of mGFP-EB3 binding affinity to E254A mutant microtubules (E254A MTs). Black symbols depict the averaged mGFP intensities measured all along the microtubules at each condition, error bars represent SD. A quadratic fit (magenta) was used to calculate the Kd. Number of microtubules measured for each mGFP-EB3 concentration: 1 nM – 96, 2.5 nM – 126, 5 nM – 86, 10 nM – 69, 20 nM – 78, 50 nM – 61, 100 nM – 64, 200 nM - 75. (j) Kymograph of mGFP-EB3 (green) single molecule imaging at 10 pM on an individual E254A microtubule (MT) in the presence of 1 nM Alexa647-EB3 (magenta). (k) Dwell time analysis of mGFP-EB3 molecules on E254A microtubule (MT) lattice plotted as a survival function (1 - CDF, cumulative density function). Dashed line (magenta) is a mono-exponential fit to the data (black dots). Number of microtubules analyzed - 126, number of mGFP-EB3 binding events - 807. Scale bars as indicated.