Position, shape, and orientation of Rtn4 oligomers. (A) An ER tubule showing the 3D surface as a cyan wireframe, the skeleton as a red line, each observed Rtn4 cluster colored uniquely, the centers of clusters as red circles, and the major axis of each cluster as a white line. (B) Polar histogram of φ for the centers of Rtn4 clusters (n = 3 cells from unique sample preparations; Error bars represent the standard deviation of the frequency of each histogram bin between biological replicates). The data represents 11,427 unique angle calculations total across the n = 3 biological replicates. (C) Similar to A but with linear-shaped simulated clusters (green) instead of observed clusters. (D) Similar to A but with circular-shaped simulated clusters (yellow) instead of observed clusters. (E) Histograms of the anisotropy of observed clusters (lavender), linear-shaped simulated clusters (green), and circular-shaped simulated clusters (yellow). (F) Similar to A excluding observed clusters and with simulated cluster major axes shown that are randomly oriented and tangential to the tubule surface. (G) Model of the vectors that ψ is being calculated between. ψ is calculated between the major axes of the clusters (magenta arrow) and vectors orthogonal to the tubule axis (blue arrow). See Materials and methods for more details. (H) Histogram of ψ for observed clusters and clusters simulated to possess all orientations tangential to the tubule surface (F). (I) Model of Rtn4 organization and its effects on tubule structure. Individual Rtn4 proteins are depicted as green cones on a magenta tubule surface.