Analysis of simulations

RMSD and RMSF calculations, ligand cluster statistics, hydrogen bond analysis, angle and distance evolutions were produced by the program Gromacs. The interval time of trajectory calculations, including RMSD, RMSF, angle, distance and principal component analysis, was 500 ps in 600 ns systems, whereas that in 100 ns 5′-GNTI systems was 100 ps. RMSD values were calculated through comparing to the initial simulation conformations. Principal components analysis was carried on through the g_covar tool. All the smooth curves in Figs 4, ​,77 and Fig. S3 were fitting groups, which were identical with corresponding Angle/RMSD calculations in color.

To determine the sub-state conformations that close ligands bound to κOR, structural clustering was performed using the g_cluster tool in Gromacs with the linkage algorithm based on the RMSD of each ligand molecule after alignment of the Cα atoms in the transmembrane helices in each frame to the starting structure⁵⁹. A 0.2 Å RMSD cut-off was chosen because it best captured spatially distinct clusters and allowed the top clusters to be representative of the predominant binding sites explored. The clustering was performed on the simulation frames with 100 ps interval time. The populations of each cluster are given in Table 1. We examined the two most populated clusters and calculated the RMSDs for each ligand found in these clusters (Table 1).

Angle analysis was carried on by g_angle tool in Gromacs. The specific input parameters were as follows. The kink angle of TM6 was measured with atom C_α of V285^6.46, P289^6.50 and F293^6.54; The angle between I294^6.55 and TM6 was measured with atom C_δ and C_α of I294^6.55 and atom C_α of W287^6.48, while that angle between E297^6.58 and TM6 was computed with atom C_δ and C_α of E297^6.58 and atom C_α of F293^6.54; The kink angle of TM7 was measured with atom C_α of Y320^7.43, P327^7.50 and A331^7.54; The angle between D105^2.50 and TM2 was measured with atom C_γ and C_α of D105^2.50 and atom C_α of I98^2.43.

NMA was conducted using the ElNemo⁶⁰ (http://www.igs.cnrs-mrs.fr/elnemo/index.html), a web interface to the elastic network model-based NMA.