The structure of CysLT1R-pran was initially solved by molecular replacement using the program Phaser (46) with two independent search models of the P2Y1R 7TM domain (PDB ID 4XNW) and BRIL from the high-resolution A2AAR structure (PDB ID 4EIY). Then, CysLT1R-zafir structure was determined by molecular replacement using 7TM domain and BRIL from the final CysLT1R-pran structure.

Model building for both CysLT1R-pran and CysLT1R-zafir structures was performed by cycling between manual inspection with Coot (47) using both 2mFo-DFc and mFo-DFc maps and automatic refinement with autoBUSTER (48) or phenix.refine (49). Restraints for pranlukast and zafirlukast were generated using the web server GRADE (version 1.2.9, available at www.globalphasing.com) and phenix.elbow. The correct orientation of the methylbenzene ring in zafirlukast (fig. S3B) was selected on the basis of the B factor and density fit comparison between two possible conformations.

In addition to zafirlukast modeled in the orthosteric binding site, an additional zafirlukast molecule was found in molecule B only, located mostly in the lipid bilayer just outside of the receptor-binding site (fig. S2, C and D). This zafirlukast molecule was likely trapped in this location due to a relatively high ligand concentration used during crystallization.

The CysLT1R-zafir structures from two molecules A and B in the asymmetric unit show very high similarity (RMSD, 0.7 Å within 7TM; RMSD, 1.1 Å for the whole structure). The main differences include a 4-Å deviation of a flexible part of ECL2 and conformations of several side chains exposed to the lipid bilayer and solvent. The final data collection and refinement statistics are shown in table S1.

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