Further information and requests for reagents should be directed to and will be fulfilled by the Lead Contact, Bryan L. Roth ( bryan_roth@med.unc.edu ).

For KOP expression, we used the Sf9 insect cells derived from the parental Spodoptera frugiperda cell line IPLB-Sf-21-AE (Expression systems). Cells were grown in ESF 921 medium (Expression systems) at 27°C and 125 rpm. Nanobodies were expressed at 27°C in E. coli WK6 (su - ) cells in TB medium (Terrific Broth, Sigma). Nanobodies were induced with 1 mM IPTG (final concentration, Isopropyl β-D-1-thiogalactopyranoside) when the bacteria density reached an OD 600 of 0.6-0.8 and bacteria cells were grown overnight at 170 rpm. For KOP functional assays, Human embryonic kidney (HEK) 293T (ATCC CRL-11268) cells were cultured in DMEM (Dulbecco’s Modified Eagle Medium). Wild-type or mutant KOP plasmids were transfected into HEK293T cells using the calcium precipitation method.

Method Details

Generation of human KOP receptor crystallization construct Wu et al., 2012 Wu H.

Wacker D.

Mileni M.

Katritch V.

Han G.W.

Vardy E.

Liu W.

Thompson A.A.

Huang X.P.

Carroll F.I.

et al. Structure of the human κ-opioid receptor in complex with JDTic. 562 RIL (BRIL) from E. coli (M7W, H102I, R106L) in place of receptor N terminus residues M1-H53, a glycine-serine linker was inserted between BRIL and receptor to facilitate crystallization. Further modifications are I135L mutation was introduced to increase expression; a haemagglutinin (HA) signal sequence followed by a FLAG tag at the N terminus, then a 10X His tag followed by a TEV protease site to enable purification by immobilized metal affinity chromatography. Crystallization of the human KOP complex was done using an engineered receptor construct that was modified based on the KOP-T4L sequence (). The final construct a) lacks N-terminal residues 1-53, b) lacks C-terminal residues 359-380, c) contains M1-L106 of the thermostabilized apocytochrome bRIL (BRIL) from E. coli (M7W, H102I, R106L) in place of receptor N terminus residues M1-H53, a glycine-serine linker was inserted between BRIL and receptor to facilitate crystallization. Further modifications are I135L mutation was introduced to increase expression; a haemagglutinin (HA) signal sequence followed by a FLAG tag at the N terminus, then a 10X His tag followed by a TEV protease site to enable purification by immobilized metal affinity chromatography.

Discovery and purification of nanobodies Pardon et al., 2014 Pardon E.

Laeremans T.

Triest S.

Rasmussen S.G.

Wohlkönig A.

Ruf A.

Muyldermans S.

Hol W.G.

Kobilka B.K.

Steyaert J. A general protocol for the generation of nanobodies for structural biology. 3.32N) bound to SalA [KOP D1383.32N was used here because it has higher affinity with SalA than wild-type ( Vardy et al., 2015 Vardy E.

Robinson J.E.

Li C.

Olsen R.H.J.

DiBerto J.F.

Giguere P.M.

Sassano F.M.

Huang X.P.

Zhu H.

Urban D.J.

et al. A new DREADD facilitates the multiplexed chemogenetic interrogation of behavior. Pardon et al., 2014 Pardon E.

Laeremans T.

Triest S.

Rasmussen S.G.

Wohlkönig A.

Ruf A.

Muyldermans S.

Hol W.G.

Kobilka B.K.

Steyaert J. A general protocol for the generation of nanobodies for structural biology. Huang et al., 2015 Huang W.

Manglik A.

Venkatakrishnan A.J.

Laeremans T.

Feinberg E.N.

Sanborn A.L.

Kato H.E.

Livingston K.E.

Thorsen T.S.

Kling R.C.

et al. Structural insights into μ-opioid receptor activation. KOP specific nanobodies were generated as described before (). In brief, one llama (Lama glama) was immunized six times with in total 0.5 mg purified BRIL-KOP DREADD (KOP D138N) bound to SalA [KOP D138N was used here because it has higher affinity with SalA than wild-type ()]. Four days after the final boost, blood was taken to isolate peripheral blood lymphocytes. RNA was purified from these lymphocytes and reverse transcribed by PCR to obtain cDNA. The resulting library was cloned into the phage display vector pMESy4 bearing a C-terminal hexa-His tag and a Glu-Pro-Glu-Ala-tag (EPEA-tag, also called or CaptureSelect C-tag). Selections were performed either on BRIL-KOP in liposomes solid phase coated directly on plates. Six different families were selected by biopanning. After two rounds of selection, periplasmic extracts were made and subjected to ELISA screens. Clones giving a positive signal in ELISA were sequenced and analyzed. Plasmids were transformed to E. coli WK6 cells, KOP specific nanobodies i.e., Nb6 and Nb7 were expressed and purified following steps 70-73 described in the previous protocol (). Nb39 DNA sequence was synthesized (Integrated DNA Technologies, IDT) based on the protein sequence in the active-state MOP structure (PDB: 5C1M ) (), and was expressed and purified using the same protocols as Nb6/7. Nanobodies were concentrated and desalted to the buffer: 10 mM HEPES, 100 mM NaCl and 10% Glycerol and stored at −80°C for future use.

Expression and purification of KOP High-titer recombinant baculovirus (> 109 viral particles per ml) was generated using the Bac-to-Bac Baculovirus Expression System (Invitrogen). ∼5 μg of recombinant bacmid in 50 μl Sf-900 II SFM media (Invitrogen) and 3 μl Cellfectin II Reagent (Invitrogen) in another 50 μl Sf-900 II SFM media (Invitrogen) were incubated for 30 min. Recombinant baculovirus was obtained by transfecting the above mixed solution into 400 μl Sf-900 II SFM media including 5x105 settled Spodoptera frugiperda (Sf9) cells (Expression Systems) in a 12-well plate (Corning). After 5 h, media was exchanged for 1 mL Sf-900 II SFM media (Invitrogen) and incubated for 5 days at 27°C. P0 viral stock with ∼109 virus particles per ml was harvested as the supernatant and used to generate high-titer baculovirus stock by infection of 40 mL of Sf9 cells (cell density: 2-3 × 106 cells/ml) and incubation for 3 days. Viral titers were determined by flow-cytometric analysis of cells stained with gp64-PE antibody (Expression Systems). Expression of KOP was carried out by infection of Sf9 cells at a cell density of 2.5 × 106 cells/ml in ESF921 media (Expression Systems) with P1 or P2 virus at a MOI (multiplicity of infection) of 3. 5% production boost additive (PBA, Expression Systems) was added to maintain cell alive. Final concentration of 10 μM naltrexone was added to help the receptor trafficking. Cells were harvested by centrifugation at 48 h post infection, washed in 1x PBS, and stored at −80°C until use. Cells were first washed by resuspending frozen cell pellets in a low-salt buffer containing 10 mM HEPES, pH 7.5, 10 mM MgCl 2 , 20 mM KCl and protease inhibitors (500 μM AEBSF, 1 μM E-64, 1 μM Leupeptin, 150 nM Aprotinin). Membranes purification was followed by 4 repeated centrifugation in a high osmolarity buffer containing 1.0 M NaCl, 10 mM HEPES, pH 7.5, 10 mM MgCl 2 , 20 mM KCl, to remove soluble and membrane associated proteins. Purified membranes were directly flash-frozen in liquid nitrogen and stored at −80°C for future use. Purified membranes were resuspended in buffer containing 10 mM HEPES, pH 7.5, 10 mM MgCl 2 , 20 mM KCl, 150 mM NaCl, 50 μM MP1104 (synthesized in house), and 1x protease inhibitors (500 μM AEBSF, 1 μM E-64, 1 μM Leupeptin, 150 nM Aprotinin), and incubated at room temperature for 1 h. The sample was then transferred to 4°C for 30 min. After another 30 min incubation in the presence of 2 mg/ml iodoacetamide (Sigma), membranes were solubilized in 10 mM HEPES, pH 7.5, 150 mM NaCl, 1% (w/v) n-dodecyl-β-D-maltopyranoside (DDM, Anatrace), 0.2% (w/v) cholesteryl hemisuccinate (CHS, Sigma), and protease inhibitors for 2 h at 4°C. The supernatant was obtained by centrifugation at 150,000 × g for 30 min and was incubated with 20 mM imidazole and TALON IMAC resin (Clontech) overnight at 4°C using approximately 500 μl resin for protein purified from 1 L of cells. The resin was then washed with 10 column volumes (cv) of Wash Buffer I (50 mM HEPES, pH 7.5, 800 mM NaCl, 0.1% (w/v) DDM, 0.02% (w/v) CHS, 20 mM imidazole, 10% (v/v) glycerol, and 25 μM MP1104, followed by 10 cv of Wash Buffer II (25 mM HEPES, pH 7.5, 150 mM NaCl, 0.05% (w/v) DDM, 0.01% (w/v) CHS, 10% (v/v) glycerol, and 25 μM MP1104). Proteins were eluted in 2.5 cv of Wash Buffer II + 250 mM imidazole, concentrated in a 100 kDa molecular weight cut-off Vivaspin 20 concentrator (Sartorius Stedim) to 500 μl, and imidazole was removed by desalting the protein over PD MiniTrap G-25 columns (GE Healthcare). The N-terminal 10 × His-tag was removed by addition of His-tagged TEV protease (Homemade) and incubation overnight at 4°C. Protease, cleaved His-tag and uncleaved protein were removed by passing the suspension through equilibrated TALON IMAC resin (Clontech) and collecting the flow-through. Excessive Nb39 (KOP/Nb39 m/m: 1:2) was then added to the protein sample and incubated for 3 h. KOP-MP1104-Nb39 complexes were then concentrated to ∼30 mg/ml with a 100 kDa molecular weight cut-off Vivaspin 500 centrifuge concentrator (Sartorius Stedim). Protein purity and monodispersity were tested by analytical size-exclusion chromatography.

Lipidic cubic phase crystallization Caffrey and Cherezov, 2009 Caffrey M.

Cherezov V. Crystallizing membrane proteins using lipidic mesophases. KOP-MP1104-Nb39 complexes were reconstituted into lipidic cubic phase (LCP) by mixing protein solution and a monoolein/cholesterol (10:1 w/w) mixture in a ratio of 2:3 v/v (protein solution/lipid) using the twin-syringe method (). Crystallization was set up in 96-well glass sandwich plates (Marienfeld GmbH) using 50 nL LCP drops dispensed from a 10 μL gas-tight syringe (Hamilton) using a handheld dispenser (Art Robbins Instruments) and overlaid with 1 μl of precipitant solution. Upon optimization, KOP-MP1104-Nb39 crystals were obtained in 100 mM Bis-tris pH 6.5-7.0, 140-200 mM magnesium sulfate hydrate, 100 mM sodium citrate tribasic dehydrate, 10 mM Manganese(II) chloride tetrahydrate, 28%–30% PEG400. Crystals grew to a maximum size of 50 μm × 30 μm × 20 μm within three days and were harvested directly from the LCP matrix using MiTeGen micromounts before flash-freezing and storage in liquid nitrogen.

cAMP inhibition assay αi -mediated cAMP inhibition, HEK293T (ATCC CRL-11268) cells were co-transfected with human KOP along with a luciferase-based cAMP biosensor (GloSensor; Promega) and assays were performed similar to previously described ( Fenalti et al., 2014 Fenalti G.

Giguere P.M.

Katritch V.

Huang X.P.

Thompson A.A.

Cherezov V.

Roth B.L.

Stevens R.C. Molecular control of δ-opioid receptor signalling. 2 overnight. The next day, drug solutions were prepared in fresh drug buffer [20 mM HEPES, 1X HBSS, 0.3% bovine serum album (BSA), pH 7.4] at 3X drug concentration. Plates were decanted and received 20 μL per well of drug buffer (20 mM HEPES, 1X HBSS) followed by addition of 10 μL of drug solution (3 wells per condition) for 15 min in the dark at room temperature. To stimulate endogenous cAMP via β adrenergic-Gs activation, 10 μL luciferin (4 mM final concentration) supplemented with isoproterenol (400 nM final concentration) were added per well. Cells were again incubated in the dark at room temperature for 15 min, and luminescence intensity was quantified using a Wallac TriLux microbeta (Perkin Elmer) luminescence counter. Results (relative luminescence units) were plotted as a function of drug concentration, normalized to % SalA stimulation, and analyzed using “log(agonist) vs. response” in GraphPad Prism 5.0. To measure KOP G-mediated cAMP inhibition, HEK293T (ATCC CRL-11268) cells were co-transfected with human KOP along with a luciferase-based cAMP biosensor (GloSensor; Promega) and assays were performed similar to previously described (). After 16 h, transfected cells were plated into Poly-lysine coated 384-well white clear bottom cell culture plates with DMEM + 1% dialysed FBS at a density of 15,000-20,000 cells per 40 μL per well and incubated at 37°C with 5% COovernight. The next day, drug solutions were prepared in fresh drug buffer [20 mM HEPES, 1X HBSS, 0.3% bovine serum album (BSA), pH 7.4] at 3X drug concentration. Plates were decanted and received 20 μL per well of drug buffer (20 mM HEPES, 1X HBSS) followed by addition of 10 μL of drug solution (3 wells per condition) for 15 min in the dark at room temperature. To stimulate endogenous cAMP via β adrenergic-Gs activation, 10 μL luciferin (4 mM final concentration) supplemented with isoproterenol (400 nM final concentration) were added per well. Cells were again incubated in the dark at room temperature for 15 min, and luminescence intensity was quantified using a Wallac TriLux microbeta (Perkin Elmer) luminescence counter. Results (relative luminescence units) were plotted as a function of drug concentration, normalized to % SalA stimulation, and analyzed using “log(agonist) vs. response” in GraphPad Prism 5.0.

Tango arrestin recruitment assay Kroeze et al., 2015 Kroeze W.K.

Sassano M.F.

Huang X.P.

Lansu K.

McCorvy J.D.

Giguère P.M.

Sciaky N.

Roth B.L. PRESTO-Tango as an open-source resource for interrogation of the druggable human GPCRome. Liu et al., 2013 Liu W.

Wacker D.

Gati C.

Han G.W.

James D.

Wang D.

Nelson G.

Weierstall U.

Katritch V.

Barty A.

et al. Serial femtosecond crystallography of G protein-coupled receptors. The KOP Tango constructs were designed and assays were performed as previously described (). HTLA cells expressing TEV fused-β-Arrestin2 (kindly provided by Dr. Richard Axel, Columbia Univ.) were transfected with the KOP Tango construct. The next day, cells were plated in DMEM supplemented with 1% dialyzed FBS in poly-L-lysine coated 384-well white clear bottom cell culture plates at a density of 10,000-15,000 cells/well in a total of 40 μl. The cells were incubated for at least 6 h before receiving drug stimulation. Drug solutions were prepared in drug buffer (20 mM HEPES, 1X HBSS, 0.3% BSA, pH 7.4) at 3X and added to cells (20 μl per well) for overnight incubation. Drug solutions used for the Tango assay were exactly the same as used for the cAMP assay. The next day, media and drug solutions were removed and 20 μl per well of BrightGlo reagent (purchased from Promega, after 1:20 dilution) was added. The plate was incubated for 20 min at room temperature in the dark before being counted using a luminescence counter. Results (relative luminescence units) were plotted as a function of drug concentration, normalized to % SalA stimulation, and analyzed using “log(agonist) vs. response” in GraphPad Prism 5.0.

GTPγ[35S] assay KOP-Gαi1, KOP-Gαi1 Y3127.35W and MOP-Gαi1 fusion constructs were transfected into HEK293T cells and membrane was prepared 48 hr later. The GTPγ[35S] assay was conducted in assay buffer (20 mM HEPES, 100 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 1 mM DTT, pH 7.4). In a 96-well plate, 20 μL of 30 μM GDP, 20 μL of 100 μM GTPγS (for Non-specific) or buffer (for Total), 20 μL of 3 nM GTPγ[35S], 20 μL of a serial dilution of KOP agonist and 120 μL of premixed membrane and 2.1mg/mL WGA-SPA PVT beads (Perkin Elmer) were added sequentially to each well (200 μL/well). The plate was sealed and agitated for 20-120 min at RT, and counted in SPA mode in a TriLux microbeta (Perkin Elmer). Results (CPM) were plotted as a function of drug concentration, normalized to % SalA or DAMGO stimulation, and analyzed using “log(agonist) vs. response” in GraphPad Prism 5.0.

Bioluminescence Resonance Energy Transfer (BRET) assay To measure KOP-nanobody recruitment, HEK293T cells were co-transfected in a 1:3 ratio with human KOP containing C-terminal Renilla luciferase (RLuc8) and nanobody containing a C-terminal YFP (with or without indicated concentrations of unlabeled Gαi1 or β-arrestin2). After at least 16 hours, transfected cells were plated in poly-lysine coated 96-well white clear bottom cell culture plates in plating media (DMEM + 1% dialyzed FBS) at a density of 40-50,000 cells in 200 μl per well and incubated overnight. The next day, media was decanted and cells were washed twice with 60 μL of drug buffer (20 mM HEPES, 1X HBSS, pH 7.4), then 60 μL of the RLuc substrate, coelenterazine h (Promega, 5 μM final concentration in drug buffer) was added per well, incubated an additional 5 minutes to allow for substrate diffusion. Afterward, 30 μL of drug (3X) in drug buffer (20 mM HEPES, 1X HBSS, 0.1% BSA, pH 7.4) was added per well and incubated for another 5 minutes. Plates were immediately read for both luminescence at 485 nm and fluorescent eYFP emission at 530 nm for 1 s per well using a Mithras LB940 multimode microplate reader. The ratio of eYFP/RLuc was calculated per well and the net BRET ratio was calculated by subtracting the eYFP/RLuc per well from the eYFP/RLuc ratio in wells without nanobody-YFP present. The net BRET ratio was plotted as a function of drug concentration using Graphpad Prism 5 (Graphpad Software Inc., San Diego, CA).

Radioligand binding and ligand dissociation assays Binding assays were performed using Sf9 membrane fractions expressing the crystallization construct BRIL-KOP or HEK293 T membrane preparations transiently expressing KOP wt or KOP mutants. Binding assays were set up in 96-well plates in the standard binding buffer (50 mM Tris, 0.1 mM EDTA, 10 mM MgCl 2 , 0.1% BSA, pH 7.40). Saturation binding assays with 0.1–20 nM [3H]-Diprenorphine or [3H]-U69,593 in standard binding buffer were performed to the determine equilibrium dissociation constant (Kd) and Bmax, whereas 10 uM final concentration of JDTic was used to define nonspecific binding. For the competition binding, 50 μL each of 3H-Diprenorphine (final 1 nM), drug solution (3X) and homogeneous membrane solution was incubated in 96-well plate in the standard binding buffer. Reactions (either saturation or competition binding) were incubated for 2 h at room temperature in the dark, and terminated by rapid vacuum filtration onto chilled 0.3% PEI-soaked GF/A filters followed by three quick washes with cold washing buffer (50 mM Tris HCl, pH 7.40) and read. Results (with or without normalization) were analyzed using GraphPad Prism 5.0 using one-site or allosteric IC 50 shift models where indicated. Radioligand dissociation assays were performed in 96-well plates in the standard binding buffer (50 mM Tris, 0.1 mM EDTA, 10 mM MgCl 2 , 0.1% BSA, pH 7.40). All assays utilized 2 concentrations of radioligand ([3H]-U69,593 = 0.5-2.0 nM) (PerkinElmer). For dissociation assays, membranes were incubated with radioligand for at least 2 hours at 37°C in the absence or presence of Nb6 or Nb39 before the addition of 10 μL of 10 μM excess cold ligand to the 200 μL membrane suspension at designated time points. Time points spanned 2 minutes to 2 hours. Non-specific binding was determined by addition of 10 μM JDTic for KOP. Immediately at time = 0 min, plates were harvested by vacuum filtration onto 0.3% polyethyleneimine pre-soaked 96-well filter mats (Perkin Elmer) using a 96-well Filtermate harvester, followed by three washes of cold wash buffer (50 mM Tris pH 7.4). Scintillation (Meltilex) cocktail (Perkin Elmer) was melted onto dried filters and radioactivity was counted using a Wallac Trilux MicroBeta counter (PerkinElmer). Data were analyzed using “Dissociation – One phase exponential decay” in Graphpad Prism 5.0.

Molecular modeling Abagyan et al., 1994 Abagyan R.

Totrov M.

Kuznetsov D. Icm - a new method for protein modeling and design - applications to docking and structure prediction from the distorted native conformation. The structure of kappa opioid receptor complex co-crystallized with MP1104 was prepared for docking experiments by addition and optimization of hydrogen atoms, and optimization of side chain residues. The ligand docking box for potential grid docking was defined as the whole extracellular half of the protein, including co-crystallized MP1104 ligand. Energy minimized structures of MP1104, IBNtxA, U-69,593, U-50,488 and Salvinorin A (SalA) were docked unto the kappa opioid receptor orthosteric site with a thoroughness value of 30, and top scored docking solutions were retained. The retained top scored docking poses were further optimized by several rounds of minimization and Monte Carlo sampling of ligand conformation and surrounding side chain residues (within 4 Å if the ligand) in the orthosteric ligand pocket. All the above molecular modeling operations were performed in ICM-Pro v3.8-5 molecular modeling package (). Van Der Spoel et al., 2005 Van Der Spoel D.

Lindahl E.

Hess B.

Groenhof G.

Mark A.E.

Berendsen H.J.C. GROMACS: fast, flexible, and free. Kim et al., 2017 Kim S.

Lee J.

Jo S.

Brooks 3rd, C.L.

Lee H.S.

Im W. CHARMM-GUI ligand reader and modeler for CHARMM force field generation of small molecules. Lee et al., 2016 Lee J.

Cheng X.

Swails J.M.

Yeom M.S.

Eastman P.K.

Lemkul J.A.

Wei S.

Buckner J.

Jeong J.C.

Qi Y.

et al. CHARMM-GUI input generator for NAMD, GROMACS, AMBER, OpenMM, and CHARMM/OpenMM simulations using the CHARMM36 additive force field. Lomize et al., 2006 Lomize M.A.

Lomize A.L.

Pogozheva I.D.

Mosberg H.I. OPM: orientations of proteins in membranes database. Best et al., 2012 Best R.B.

Zhu X.

Shim J.

Lopes P.E.M.

Mittal J.

Feig M.

Mackerell Jr., A.D. Optimization of the additive CHARMM all-atom protein force field targeting improved sampling of the backbone φ, ψ and side-chain χ(1) and χ(2) dihedral angles. Krivov et al., 2009 Krivov G.G.

Shapovalov M.V.

Dunbrack Jr., R.L. Improved prediction of protein side-chain conformations with SCWRL4. −1 or 100,000 iterations). This was followed by a HINT ( Eugene Kellogg and Abraham, 2000 Eugene Kellogg G.

Abraham D.J. Hydrophobicity: is LogP(o/w) more than the sum of its parts?. Laskowski et al., 1993 Laskowski R.A.

MacArthur M.W.

Moss D.S.

Thornton J.M. PROCHECK: a program to check the stereochemical quality of protein structures. For SalA, an additional round of simulations was performed by molecular dynamics (MD) methods. Gromacs 5.0.4 () was used to perform all MD simulations. All input files for MD simulation of the docked conformation of SalA and kappa opioid receptor complex, and parameter files for SalA were generated using CHARMM-GUI server (). The orientation of helices within the membrane was derived after overlapping the complex with orientation of 4DJH (the JDTic co-crystallized kappa opioid receptor structure) obtained from the OPM server (). Two MD runs of membrane embedded and water boxed SalA-kappa opioid receptor complex (including 218 POPC lipid molecules, 19206 water molecules, 56 sodium ions and 61 chloride ions) were simulated with the CHARMM forcefield () at 310K temperature with a step size of 2 femtoseconds using 6 GPU-enabled nodes with 16 processors for a period of 650 ns and 500 ns, after minimization and equilibrations. During the MD runs the hydrogen atoms were constrained using LINCS and cut-off of 12 Å was used for Van der Waals and short range electrostatic interactions, along with PME conditions. MD-derived receptor–ligand complexes were then subjected to an iterative refinement process guided by experimental data from mutagenesis studies and SalA structure–activity relationships (SAR). In the first step, rotatable bonds of the ligand and/or amino acid side chains of KOP were modified either manually (for the ligand) or algorithmically with a rotamer library using SCWRL4 () (for KOP) so as to maximize the stereoelectronic complementarity of the interacting partners. An energy minimization step was then performed on the complex in SYBYL-X 2.1.1 (Certara USA, Inc., Princeton, NJ) using the Tripos Force Field (Gasteiger−Hückel charges, distance-dependent dielectric constant = 4.0 D/Å, termination criteria: energy gradient cut-off = 0.05 kcal (mol × Å)or 100,000 iterations). This was followed by a HINT () analysis to assess the energetic favorability of the receptor–ligand complex. The iterative refinement process terminated when no further energetically favorable structural modifications could be identified. The stereochemical quality of the final models was assessed using PROCHECK ().