X-ray Diffraction of NK1R-Ligand Complexes

X-ray diffraction data were collected at the X06SA beamline at the Swiss Light Source (SLS) of the Paul Scherrer Institute (PSI, Villigen, Switzerland) using a beam size of 10 × 10 μm and an EIGER 16 M detector. Datasets for CP-99,994-bound NK₁R were collected using a beam attenuated to 10%, 0.1° of oscillation and 0.1 s exposure time. All other datasets were collected using a beam attenuated to 30%, 0.1° of oscillation and 0.05 s exposure time. Data from individual crystals were integrated using XDS^{66 (link)}. Data merging and scaling was carried out using the program AIMLESS from the CCP4 suite^{67 (link),68 (link)}. Data collection statistics are reported in Table 1.
Initial phases were obtained by molecular replacement (MR) with the program Phaser^{69 (link)} using the truncated OX2R transmembrane domain (PDB ID 4S0V) and the separated PGS fusion protein^{31 (link)} as independent search models looking for one copy of each domain. Manual model building was performed in COOT^{70 (link)} using sigma-A weighted 2m|F_o|-|DF_c|, m|F_o|-D|F_c| maps together with simulated-annealing and simple composite omit maps calculated using Phenix⁷¹ . Initial refinement was carried out with REFMAC5^{72 (link)} using maximum-likelihood restrained refinement in combination with the jelly-body protocol. Further and final stages of refinement were performed with Phenix.refine^{73 (link)} with positional, individual isotropic B-factor refinement and TLS. The final refinement statistics are presented in Table 1. Co-ordinates and structure factors have been deposited in the worldwide Protein Data Bank under accession codes 6HLL, 6HLO and 6HLP for the CP-99,994-, aprepitant- and netupitant-bound NK₁R, respectively.