(A) Simulation rules and parameters: (1) EB1 diffuses in three dimensions, (2) EB1 is obstructed by tubulin dimers and cannot pass through microtubules, (3) EB1 and tubulin dimer three-dimensional shapes reproduce EB1-tubulin binding interface. (B) Development of simulation shapes for EB1 and tubulin: Left: Cryo-EM reconstruction data (from PDB ID 3JAR) of tubulin dimers in a lattice: Left-top: Top-down view, with the lower portion being the outside of the microtubule. Left-bottom; Side view of four tubulin dimers, viewing the portion on the outside surface of the microtubule; Middle: Approximation of tubulin dimers for use in the simulation, derived from the cryo structure; Right: EB1 (red) also modeled as an approximation of cryo-structure data (not shown), with the binding interface correctly positioned at the pocket located between four adjacent tubulin dimers. (C) Two microtubule conditions used in the simulation analysis. Left: A closed, blunt-ended microtubule. Right: A ‘disrupted-structure’ microtubule with a tapered tip. Both the ‘closed’ and ‘disrupted structure’ configurations contain the same total number of tubulin dimers. (D) Results of 50 simulations of 4000 EB1 molecules each for each microtubule condition. Each data point in the box and whisker plot represents the total number of EB1 arrival events per microtubule for 4000 different simulated EB1 molecules. (E) Visualization of EB1 arrival events (red) on closed and ‘disrupted-structure’ microtubules from N = 10,000 simulated EB1 in each condition. (F) Illustration of the hypothesis that EB1’s pocket-located binding site leads to a high local steric hindrance for EB1 binding to a lattice conformation (top), and a lower steric hindrance for EB1 binding to an edge conformation (bottom). Green lines: portion of the local volume with high accessibility to binding site. Yellow dotted lines: reduced accessibility volume. Red dashed lines: volume with no direct accessibility. In an edge conformation (bottom), the high accessibility region (green) is much larger than in the lattice conformation (top). (G) Dark Red arrows: Binding sites with two adjacent tubulin dimers, such as at the ends of protofilaments, or at exposed edges of protofilaments with no neighbor, are termed ‘Edge’ sites. Light red arrow: Sites with four adjacent tubulin dimers are termed ‘Lattice’ sites. Sites with only one tubulin dimer and with three adjacent tubulin dimers were not included in this analysis. (H) Fraction of simulated EB1 molecules that arrived at lattice sites (light red) or at edge sites (dark red). Data used was from the closed microtubule arrangement (panel C,E, left)). Values were determined by dividing the total number of EB1 bound at any lattice or edge site by the total number of available lattice or edge sites, respectively. EB1 was ~70 fold more likely to bind a given edge site as compared to a given lattice site. (I) Left: example electron microscopy images of EB1 conjugated to gold beads on GDP microtubules. This image shows EB1-gold bound at a sheet edge, and one bound at a sheet middle (beads at ambiguous locations were conservatively classified as ‘middle’). Right: Count of total number of sheet bound beads observed over all images, divided into ‘Middle’ of sheet and ‘Edge’ of sheet. (J) Far Left Top; Experimental setup, rhodamine-labeled microtubules are affixed to a coverslip (red), EB1-GFP is introduced in solution (green), and the sample imaged at 100 frames per second using total internal reflection fluorescence (TIRF) microscopy. Far Left Bottom; Sample image of EB1-GFP on the microtubule. Left: Kymograph of EB1-GFP with length along the x-axis and time down the y-axis. White arrows indicate EB1-GFP binding events, which appear as vertical streaks. Example shows atypically long EB1 association events, for clarity. The lower limit of the vertical streaks are the dissociation event of EB1-GFP from the microtubule. Right: EB1-GFP on-rate constant for closed and disrupted-structure microtubules in each nucleotide population.