Effects of a larger membrane protein on the density profiles and thickness measurements in MD-derived EM volumes. (A) Side view (XZ) of a tetrameric TRPV4 core (281.6 kDa, lilac) in a symmetric phosphatidylcholine (POPC) bilayer by Goretzki et al. (2023), converted to an EM density map (see Materials and methods). Lipid tails are shown as gray lines, and phosphate headgroups as yellow spheres. Water molecules are hidden for clarity. (B) Top view (XY) of the same simulation system. (C) Map of the distance-dependent analysis. Annotated: protein center of mass (COM, black point), convex hull footprint of the protein on the membrane (blue irregular outline), equivalent radius circle (dashed blue), and color-coded annular rings of extracted membrane regions at different distances from the protein center. (D) Density profiles per annular ring. Annotated: hydrophobic core maximum (gray point), headgroup density minima (light pink points), phosphate atom positions (yellow points), and profile inflection points (density gradient extrema, dark purple points). The isolated protein density profile is shown at the bottom for comparison. (E) Example density gradient profile (1–2 nm distance bin). Gradient extrema (inflection points on the profiles in D) are indicated by dark purple points. (F) Comparative membrane thickness measurements using minima-to-minima (light bars) distances vs distances between inflection points (hatched bars, described in Materials and methods). Thickness measured by both approaches is smaller compared with the simulated asymmetric PM-like system in Fig. 3 F.