Orientation-dependent analysis of membrane thickness measurements for the ER. (A) Central slices from four representative of C. reinhardtii tomograms containing ER membranes in different orientations (Kelley et al., 2024, Preprint) (EMPIAR-11830, 7.84 Å/pixel at bin4, 302.mrc, 2131.mrc, 2268.mrc, and 2161.mrc). Scale bars, 100 nm. (B) MemBrain (Lamm et al., 2025, Preprint) segmentations of the corresponding ER membranes, color-coded to match the distribution plots in C. (C) Histogram distributions of membrane orientation angles relative to the missing wedge (0° = parallel, 90° = perpendicular to missing wedge), calculated using dot products between surface point normal vectors and the z-axis unit vector (see Materials and methods). The Pearson correlation coefficients between membrane thickness and orientation angle are indicated in each dataset’s legend. (D) Membrane thickness measurement distributions for the four ER instances. (E) Tomogram mean defocus values.