Methods
A total of 216 GPCR structures (119 annotated as active and 97 as inactive) were downloaded from the GPCRdb12, selecting all TMD helices, helix 8 and the loops between these elements. Protons were added using PyMol v2.4.213. PDB identification codes of the structures are listed in table S1 in the supplementary information (SI). Packing densities were calculated by counting the number of atoms within a 5 Å radius around the δ-methyl carbons of Leu and Ile. Atoms that belong to the same residue as the probed methyl group were not included in the count. The area of side chains at the protein surface was quantified by determining the solvent-accessible surface areas using GETAREA14 with default settings including a water probe radius of 1.4 Å. The relative area of side chains at the protein surfaces was calculated based on mean values obtained for side chains of the free amino acids (174.2 Å2 for Ile and 174.0 Å2 for Leu).
Class A GPCR sequences (1580 in total from 325 targets) were downloaded from GPCRdb selecting only sequences of TMD helices. Hydropathies were calculated based on the differences of Wimley-White whole-residue hydrophobicity scales for the transfer of an amino acid from water to a bilayer interface and from water to octanol (ΔGwoct-ΔGwif).10,11The sum of all amino acid hydropathies was taken as the hydropathy of the TMDs. Only the hydropathies for protonation states of amino acids at pH 7 were considered. Spearman’s rank correlation coefficients (ρ) were calculated based on amino acid content, i.e. the number of residues of an amino acid within the TMD divided by the sequence length of the TMD. All calculations, statistical analyses, and plots were done using R v4.0.315 with RStudio v1.4.110316and the packages bio3d17 and stringr18.