Supplementary Materials

Supplementary Materials and Methods Homology modeling and docking. Homology models of mOR256-3, mOR256-8 and mOR256-31 were built using the approach in ref. 1,2. Four X-ray crystal structures of class A GPCRs were used as templates, (1U19), CXCR4 (3ODU), A2aR (2YDV) and CXCR1 (2LNL), to build 100 models with Modeller v9.15 3. For docking, we chose the model with the lowest DOPE score. Autodock Vina 4 and the Haddock 2.2 webserver 5 were used to identify a common top-ranked binding pose for each odorant. Residues in the putative ligand-binding pocket were set flexible during docking. Molecular dynamics. The -odorant complexes were embedded in a bilayer of POPC using Desmond- Maestro (v2016.1, non-commercial distribution) 6. Each system was solvated in a periodic 75 × 75 × 105 Å3 box of explicit and neutralized with 0.15 M of Na+ and Cl- ions. Effective point charges of the ligands were obtained by RESP fitting 7 of the electrostatic potentials calculated with the HF/6-31G* basis set using Gaussian 09 8. The Amber 99SB-ildn 9, lipid 14 10 and GAFF 11 force fields were used for the , the lipids and the ligands, respectively. The TIP3P 12 and the Joung-Cheatham 13 models were used for the water and the ions, respectively. After energy minimization, all-atom MD simulations were carried out using Gromacs 5.1 patched with the PLUMED 2.3 plugin 14. Each system was gradually heated to 310 K and pre-equilibrated during 10 ns of brute-force MD in the NPT-ensemble (see SI-Methods for details). The replica exchange with solute scaling (REST2) 15 technique was then employed to enhance the sampling with 48 replicas in the NVT ensemble. The and the ligands were considered as “solute” in the REST2 scheme–force constants of their van der Waals, electrostatic and dihedral terms were subject to scaling. The effective temperatures used for generating the REST2 scaling factors ranged from 310 K to 700 K, following a distribution calculated with the Patriksson-van der Spoel approach 16. Exchange between replicas was attempted every 1000 simulation steps. This setup resulted in an average exchange probability of ~40%. The original unscaled replica (at 310 K effective temperature) was collected and analyzed. The first 10 ns were discarded for equilibration. Cluster analysis of the ligand binding pose was carried out on the non-restrained trajectory using g_cluster in Gromacs tools with the GROMOS method 17. The middle structure of the most populated cluster was selected as the final binding pose.

Fig. S1. Dose-dependent response of mOR256-31 mutants to coumarin and R-carvone. The residues were mutated to the corresponding ones in mOR256-8 (red), a narrowly tuned OR that does not response to R-carvone or coumarin 18. Four other mutations (blue and green) were made to Y1043.32, G1083.36 and V1995.39.

Fig. S2. Matthew’s correlation coefficient (MCC) as metric of RF classifier predictivity, tested on the external test set of 43 ORs. Labeled in parenthesis is the number of optimal residues that gave the best performance in each model. The best models are highlighted in red. The control models (random) were built with randomly chosen residues (same number of residues as in the best model).

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Figure S3. Residue positions that, upon mutation, significantly altered the basal activity of class A GPCRs (Table S8).

Fig. S4. RPART classifiers’ performance on OR basal activity. (A) The training set during 5-fold cross validation repeated 5 times. (B) The external test set repeated 5 times. Labeled in parenthesis is the number of optimal residues that gave the best performance in each model. The best models are highlighted in red. The control models (random) were built with randomly chosen residues (same number of residues as in the best model).

Figure S5. Current knowledge of the combinatorial codes for the 4 odorants illustrated on the phylogenetic tree of hORs. The large to small hOR label sizes indicate the strength of response (EC50 < 10 µM, 10 µM < EC50 < 100 µM and EC50 > 100 µM, respectively). hORs identified in this work are labelled in blue.

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Table S1. Deorphanized human and mouse ORs to date.

Count Refs hOR10A5, hOR10A6, hOR10A7, hOR10AG1, hOR10C1, hOR10G3, hOR10G4, hOR10G6, hOR10G7, hOR10H1, hOR10J3, hOR10J5, hOR10K1, hOR11A1, hOR11G2, hOR11H1, hOR11H13, hOR11H2, hOR11H4, hOR11H6, hOR11H7, hOR13C2, hOR13C5, hOR13C8, hOR13C9, hOR13G1, hOR14C36, hOR14J1, hOR14L1, hOR1A1, hOR1A2, hOR1C1, hOR1D2, hOR1D5, hOR1E1, hOR1E3, hOR1G1, hOR1J1, hOR1J4, hOR1L3, hOR1N1, hOR1N2, hOR1S1, hOR1S2, hOR2A25, hOR2A4, hOR2A7, hOR2AG1, hOR2AK2, hOR2AT4, hOR2B11, hOR2B2, hOR2B3, hOR2C1, hOR2C3, hOR2D2, hOR2F1, hOR2G2, hOR2J2, hOR2J3, hOR2L2, hOR2L5, hOR2L8, hOR2M3, hOR2M4, hOR2M7, hOR2T1, hOR2T10, hOR2T11, hOR2T29, hOR2T34, hOR2T35, hOR2T6, hOR2T8, hOR2V2, hOR2W1, hOR2Y1, hOR3A1, hOR3A3, hOR3A4, hOR4A16, hOR4A4, hOR4A47, Human hOR4A5, hOR4D1, hOR4D10, hOR4D2, hOR4D6, hOR4D9, hOR4E2, hOR4F17, 180 19-34 ORs hOR4F4, hOR4K1, hOR4K14, hOR4K15, hOR4K5, hOR4M1, hOR4N2, hOR4N5, hOR4P4, hOR4Q3, hOR4X2, hOR51A2, hOR51B2, hOR51B4, hOR51B5, hOR51B6, hOR51D1, hOR51E1, hOR51E2, hOR51F1, hOR51I2, hOR51J1, hOR51L1, hOR51M1, hOR51S1, hOR51T1, hOR51V1, hOR52A5, hOR52D1, hOR52E5, hOR52E8, hOR52H1, hOR52I1, hOR52J3, hOR52K1, hOR52K2, hOR52L1, hOR52M1, hOR52N1, hOR52N2, hOR52N5, hOR52R1, hOR52W1, hOR56A3, hOR56A4, hOR56A5, hOR56B4, hOR5A1, hOR5A2, hOR5AK2, hOR5AL1, hOR5AN1, hOR5AS1, hOR5B12, hOR5B17, hOR5B3, hOR5C2, hOR5D14, hOR5D18, hOR5H2, hOR5J2, hOR5K1, hOR5L1, hOR5M11, hOR5P2, hOR5P3, hOR6C1, hOR6C65, hOR6C68, hOR6J1, hOR6K2, hOR6P1, hOR6S1, hOR7A5, hOR7C1, hOR7D4, hOR7G2, hOR8B12, hOR8B2, hOR8B3, hOR8D1, hOR8G5, hOR8J1, hOR8K3, hOR9A2, hOR9G1, hOR9G4, hOR9Q1, hOR9Q2 Olfr64, Olfr1019, Olfr1032, Olfr1062, Olfr1062, Olfr1079, Olfr1079, Olfr109, Olfr109, Olfr1093, Olfr1104, Olfr1104, Olfr124, Olfr1264, Olfr1324, Olfr1325, Olfr1328, Olfr1341, Olfr1352, Olfr1356, Olfr1364, Olfr1370, Olfr1377, Olfr1377, Olfr1395, Olfr1411, Olfr1413, Olfr145, Olfr1484, Olfr15, Olfr1509, Olfr151, Olfr1512, Olfr154, Olfr16, Olfr16, Olfr160, Olfr161, Olfr167, Olfr167, Olfr168, Olfr171, Olfr175, Olfr19,

Olfr2, Olfr202, Olfr211, Olfr214, Olfr221, Olfr24, Olfr311, Olfr320, Olfr323, Olfr340, 19,21, Mouse Olfr406, Olfr429, Olfr447, Olfr459, Olfr476, Olfr480, Olfr49, Olfr491, Olfr50, 124 22,33, ORs Olfr502, Olfr508, Olfr510, Olfr514, Olfr532, Olfr544, Olfr545, Olfr547, Olfr549, 35-60 Olfr554, Olfr556, Olfr556, Olfr558, Olfr56, Olfr569, Olfr599, Olfr609, Olfr61, Olfr611, Olfr616, Olfr62, Olfr620, Olfr638, Olfr642, Olfr644, Olfr65, Olfr653, Olfr661, Olfr67, Olfr672, Olfr677, Olfr678, Olfr68, Olfr683, Olfr685, Olfr690, Olfr691, Olfr715, Olfr73, Olfr74, Olfr744, Olfr749, Olfr749, Olfr790, Olfr796, Olfr876, Olfr876, Olfr889, Olfr895, Olfr90, Olfr909, Olfr919, Olfr961, Olfr963, Olfr978, Olfr979, Olfr979, Olfr982, Olfr983, Olfr992, Olfr992

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Table S2. OR-odorant pairs used in machine learning model training and cross validation. Responsive Non-responsive Ref hOR10G7, hOR2W1, hOR5P3, Olfr1009, Olfr1044, Olfr1047, Olfr1054, Olfr1062, Olfr1079, Olfr109, hOR10A3, hOR10A7, hOR10C1, hOR10J5, hOR10K1, hOR10Q1, hOR10R2, hOR10X1, Olfr1093, Olfr1094, Olfr110, Olfr1104, Olfr1126, hOR12D2, hOR13D1, hOR14J1, hOR14L1, hOR1C1, hOR1D5, hOR1E2, hOR1G1, Olfr1170, Olfr1238, Olfr124, Olfr133, Olfr1333, hOR1J2, hOR1L8, hOR1N2, hOR2A12, hOR2A4, hOR2AG1, hOR2AT4, hOR2G6, Olfr1352, Olfr136, Olfr1370, Olfr1377, Olfr143, hOR2L13, hOR2L2, hOR2L3, hOR2L8, hOR2M4, hOR2M7, hOR2T5, hOR2W5, Olfr1443, Olfr1444, Olfr1448, Olfr145, Olfr1463, hOR4A16, hOR4C15, hOR4C3, hOR4C5, hOR4F16, hOR4M2, hOR4N4, hOR4S1, Olfr1469, Olfr1484, Olfr151, Olfr156, Olfr160, hOR51E1, hOR51E2, hOR51F1, hOR51L1, hOR52B2, hOR52D1, hOR52I1, hOR56A1, 21,61 acetophenone Olfr166, Olfr167, Olfr168, Olfr191, Olfr196, Olfr201, hOR5AL1, hOR5D16, hOR5H1, hOR5H2, hOR5H6, hOR5M9, hOR5P2, hOR5T1, -63 Olfr203, Olfr205, Olfr30, Olfr339, Olfr346, Olfr347, hOR6C3, hOR6C6, hOR6C65, hOR6C75, hOR6F1, hOR6K3, hOR6K6, hOR6N2, Olfr376, Olfr414, Olfr429, Olfr430, Olfr434, Olfr466, hOR7A10, hOR7A5, hOR7G2, hOR8A1, hOR8B8, hOR8G5, hOR9A2, hOR9Q2, Olfr476, Olfr478, Olfr490, Olfr491, Olfr494, Olfr502, Olfr1019, Olfr1264, Olfr1324, Olfr1395, Olfr15, Olfr171, Olfr174, Olfr19, Olfr202, Olfr51, Olfr556, Olfr57, Olfr60, Olfr62, Olfr736, Olfr221, Olfr311, Olfr323, Olfr340, Olfr508, Olfr532, Olfr554, Olfr558, Olfr569, Olfr739, Olfr744, Olfr746, Olfr749, Olfr874, Olfr875, Olfr599, Olfr609, Olfr61, Olfr611, Olfr632, Olfr638, Olfr64, Olfr653, Olfr67, Olfr683, Olfr876, Olfr887, Olfr888, Olfr889, Olfr890, Olfr895, Olfr685, Olfr715, Olfr796, Olfr979, Olfr992 Olfr904, Olfr920, Olfr923, Olfr935, Olfr983 hOR10A2, hOR10A5, hOR10A6, hOR10A7, hOR10AG1, hOR10C1, hOR10G6, hOR10G7, hOR10K2, hOR10Q1, hOR10X1, hOR11H1, hOR12D3, hOR13C2, hOR13C4, hOR13C8, hOR13D1, hOR14A2, hOR1C1, hOR1D5, hOR1E2, hOR1G1, hOR1J1, hOR1L8, hOR1N1, hOR1N2, hOR1S1, hOR1S2, hOR2A1, hOR2A12, hOR2AG1, hOR2AT4, hOR2B2, hOR2C1, hOR2D2, hOR2F1, hOR2G6, hOR2J2, hOR2K2, hOR2L2, hOR2L5, hOR2L8, hOR2M3, hOR2M7, hOR2T29, hOR2T5, hOR2T8, hOR2Z1, hOR4A16, hOR4A5, hOR4B1, hOR4C11, hOR4C15, hOR4C3, hOR1A1, hOR2W1, hOR2Y1, hOR5P3, hOR6X1, hOR4D10, hOR4D5, hOR4F16, hOR4F17, hOR4F4, hOR4F5, hOR4K13, hOR4K2, 20,21 Olfr1079, Olfr124, Olfr1352, Olfr1356, Olfr168, hOR4K3, hOR4K5, hOR4M1, hOR4N2, hOR4N4, hOR4N5, hOR4S1, hOR4X2, R-carvone ,35,6 Olfr174, Olfr263, Olfr340, Olfr362, Olfr45, Olfr50, hOR5A2, hOR5AK3, hOR5AN1, hOR5AR1, hOR5B12, hOR5B17, hOR5D16, hOR5H6, 3,64 Olfr556, Olfr895, Olfr992 hOR5L1, hOR5M11, hOR5M9, hOR5T2, hOR5V1, hOR6A2, hOR6C65, hOR6C75, hOR6F1, hOR6K6, hOR6M1, hOR6P1, hOR7A5, hOR7C1, hOR7D4, hOR7G1, hOR7G2, hOR7G3, hOR8A1, hOR8B12, hOR8K3, hOR8G5, hOR8U8, hOR9G1, hOR9Q2, Olfr1019, Olfr1062, Olfr109, Olfr1104, Olfr1264, Olfr1324, Olfr1341, Olfr1377, Olfr1395, Olfr15, Olfr171, Olfr19, Olfr202, Olfr221, Olfr311, Olfr323, Olfr429, Olfr532, Olfr554, Olfr558, Olfr569, Olfr599, Olfr609, Olfr61, Olfr611, Olfr632, Olfr638, Olfr64, Olfr653, Olfr67, Olfr683, Olfr685, Olfr715, Olfr749, Olfr796, Olfr979, Olfr983 hOR1A1, hOR10J5, hOR2C1, hOR2M7, Olfr167, Olfr168, Olfr992, Olfr15, Olfr171, hOR1C1, hOR2B11, hOR2J2, hOR2W1, hOR5P3, Olfr340, Olfr429, Olfr715, Olfr1395, Olfr1324, Olfr323, Olfr1264, Olfr979, Olfr796, 18,19 Olfr1062, Olfr109, Olfr1104, Olfr124, Olfr1328, coumarin Olfr174, Olfr202, Olfr61, Olfr532, Olfr311, Olfr1019, Olfr221, Olfr19, Olfr447, Olfr514, ,21,6 Olfr1341, Olfr1352, Olfr1377, Olfr263, Olfr508, Olfr64, Olfr632, Olfr569, Olfr685, Olfr609, Olfr611, Olfr683, Olfr653, Olfr638, Olfr554, 3 Olfr556, Olfr749, Olfr876, Olfr895, Olfr983 Olfr67, Olfr599, Olfr558 hOR5P3, hOR2J2, hOR10J5, hOR2C1, hOR2M7, Olfr749, Olfr109, Olfr508, Olfr1341, hOR1A1, hOR2W1, Olfr1062, Olfr1079, Olfr1104, Olfr992, Olfr15, Olfr429, Olfr715, Olfr1324, Olfr323, Olfr1264, Olfr979, Olfr796, 4-chromanone Olfr1352, Olfr1377, Olfr1395, Olfr167, Olfr168, Olfr174, Olfr202, Olfr61, Olfr532, Olfr1019, Olfr221, Olfr19, Olfr447, Olfr514, Olfr64, 21 Olfr171, Olfr311, Olfr340, Olfr556, Olfr895, Olfr983 Olfr632, Olfr569, Olfr685, Olfr609, Olfr611, Olfr683, Olfr653, Olfr638, Olfr554, Olfr67, Olfr599, Olfr558

Table S3. mOR256-31 residues in direct contact with the odorants. Ballesteros- Occupation (%) during MD a Literature data (mutagenesis) Weinstein Coumarin R-carvone Acetophenone Receptor b Refs. nomenclature 2.53 100 38 44 3.29 0 72 49 hOR1A1 3.30 0 17 0 mOR256-3, mOR256-8 3.32 100 100 96 hOR1A1, hOR2AG1, mOR-EG, mOR256-3 3.33 100 100 100 hOR1A1, mOR-EG, mOR42-3 hOR1A1, hOR1A2, mOR-EG, mOR42-3, 3.36 100 99 99 mOR256-3 3.37 9 81 97 hOR1A1, hOR1A2, mOR42-3, mOR244-3

3.38 0 11 1 1,2,18,35 5.42 40 92 99 hOR1A1, mOR-EG, mOR42-3 ,39,65-69 5.43 0 74 99 mOR42-3, mOR256-3 hOR1A1, hOR1A2, hOR2AG1, mOR-EG, 5.46 94 71 59 mOR42-3 5.47 0 89 97 hOR1A1, mOR-EG 6.48 100 85 97 hOR1A1, hOR7D4, mOR256-3 6.51 100 100 73 hOR1A1, hOR1A2, mOR-EG, mOR42-3 6.52 0 96 100 6.55 0 97 97 mOR-EG 7.42 100 30 1 hOR1A1, hOR1A2, hOR2AG1, mOR-EG a Percentage of the simulation trajectory where the residue was within 5 Å distance of the odorants. b Receptor response to odorants was affected upon mutation.

Table S4. Six subsets of residues tested in machine learning of OR response to odorants.

Subset Number of residues Ballesteros-Weinstein nomenclature poc-17 17 2.53 3.29 3.30 3.32 3.33 3.36 3.37 3.38 5.42 5.43 5.46 5.47 6.48 6.51 6.52 6.55 7.42 poc-20 20 2.53 2.54 3.29 3.30 3.32 3.33 3.36 3.37 3.38 5.38 5.39 5.42 5.43 5.46 5.47 6.48 6.51 6.52 6.55 7.42 poc-27 27 2.53 2.54 2.57 3.29 3.30 3.31 3.32 3.33 3.35 3.36 3.37 3.38 4.48 4.52 5.38 5.39 5.41 5.42 5.43 5.46 5.47 6.48 6.51 6.52 6.55 7.39 7.42 poc-60 60 2.53 2.54 2.57 2.61 3.25 3.26 3.27 3.28 3.29 3.30 3.31 3.32 3.33 3.34 3.35 3.36 3.37 3.38 3.39 3.40 3.41 4.52 4.53 4.56 4.57 4.60 5.35 5.36 5.37 5.38 5.39 5.40 5.41 5.42 5.43 5.44 5.45 5.46 5.47 5.48 5.49 5.50 5.51 6.44 6.45 6.47 6.48 6.49 6.50 6.51 6.52 6.53 6.54 6.55 6.56 7.35 7.38 7.39 7.41 7.42 7TM-191 191 1.32–1.65, 2.39–2.67, 3.25–3.55, 4.40–4.61, 5.37–5.65, 6.32–6.56, 7.35– 7.55 7TM-ECL2-214 214 Subset V + 23 residues between the 2 conserved cysteines in ECl2

Table S5. Newly identified OR-odorant pairs and EC50 (with 95% confidence interval) a in Hana3A cells.

acetophenone R-carvone coumarin 4-chromanone hOR11H12 4.7 (0.7–29.9) 16.3 (5.5–48.4) 91.9 (16.6–506.7) n.r. hOR5R1 3.4 (0.9–12.7) 12 (3.2–32.7) 10.2 (2.2–64.3) 217.8 (10.5–4506) hOR8B2 n.r. n.r. n.r. 15 (2.2–101.4) hOR8K5 n.r. 4.5 (1.0–21.3) n.r. n.r. Olfr1097 57.3 (14.5–226.8) n.r. 7.2 (1.0–54.2) 9.5 (1.8–49.5) Olfr1016 n.r. n.r. 72.4 (14.3–336.1) n.r. Olfr1057 n.r. n.r. 3.8 (1.3–11.5) 5.8 (2.6–13.2) Olfr1156 46.2 (9.6–222.8) 30.5 (1.2–789) n.r. n.r. Olfr285 n.r. n.r. 66.7 (32.3–137.8) 72.8 (14.5–366.7) Olfr924 n.r. n.r. 28 (4.5–173) n.r. a n.r. indicates no significant response up to 1 mM.

Table S6. Residue positions that, upon mutation, significantly altered the basal activity of class A GPCRs.

Receptor Gain of function Loss of function Ref. A1b 3.49, 3.50, 6.30, 6.34 70,71 Adenosine 1a receptor 6.34 71 Adenosine 2a receptor 3.39 4.46, 4.49, 4.56, 4.58, 4.60, 72,73 4.61, 5.35, 5.38, 5.42, 5.40, 5.47 Angiotensin II type 1 receptor 3.35, 3.41, 4.49 74,75 B2 adrenergic receptor 3.49, 6.30, 6.44, 6.47, 7.46 76-78 B3 adrenergic receptor 5.47, 6.51, 6.52 79 Canabinoid receptor 1 2.37, 2.63, 3.43 80,81 C-C 5 2.65 82 Cholecystokinin B receptor 3.32, 5.40, 5.52 83 C-X-C chemokin receptors 1–4 2.53, 2.56, 3.35, 6.40 84-86 δ- 3.32, 7.42, 8.53 87,88 Follicle-stimulating hormone 3.32, 5.54, 6.30 89 receptor GPR18 3.39 90 Growth hormone secretagogue 3.40, 3.43 6.48 91 receptor Gonadotropin-releasing 2.53, 6.40, 6.47, 6.52 92,93 Histamine 1 receptor 6.40 94 Luteinizing hormone receptor 1.41, 1.46, 2.43, 3.43, 5.54, 6.30, 2.50, 5.58 89,95 6.34, 6.37, 6.38, 6.41, 6.43, 6.44, 6.47, 6.48 µ-opioid receptor 3.35, 3.49 96,97 Muscarinic receptors 1–5 6.58, 6.59 98 1 7.42 99 3.35 100 Parathyroid hormone 1 receptor 2.43, 6.37, 7.52 101 receptor 3.50 102 Rhodopsin 2.46, 2.57, 2.61, 3.28, 6.40, 7.38, 89,103-107 7.39, 7.42, 7.43, 7.54 Serotonin 5HT4 receptor 2.50, 3.36, 6.48 108,109 Serotonin 5HT2a receptor 6.34 110 1.39, 1.49, 2.43, 2.53, 2.56, 2.58, 2.59, 2.60, 5.50, 6.47 89,101,111- 3.32, 3.36, 3.43, 3.40, 5.44, 5.54, 114 5.58, 6.30, 6.34, 6.37, 6.40, 6.42, 6.43, 6.44, 6.48, 6.50, 6.52, 6.54, 6.56, 6.59, 7.42, 7.45, 7.47, 7.52 receptor 2 3.43, 3.50, 5.62 101

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Supporting references

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