Individual diffraction datasets were analyzed using XDS (45) for integrating Bragg peaks. All datasets showed the same unit cell dimensions with negligible deviation (less than 0.5%) and space group p61. The best five datasets were chosen for merging together to improve the signal-to-noise ratio. The datasets were combined using XSCALE without scaling and merging, and the pooled reflection list was further analyzed using the STARANISO server (Global Phasing Ltd.). The STARANISO server first analyzed the anisotropy for each dataset, giving a resolution of 3.69 Å in the hk plane and 3.11 Å in the l direction using the criterion of CC1/2 = 0.3. The scaling factor was determined and applied after applying the anisotropic mask. After scaling, the datasets were merged, and the final resolution was improved to 3.02 Å and anisotropically truncated to 3.12 Å in the best direction (table S1). Structures of rhodopsin (PDB ID: 4A4M) and mini-Gs (PDB ID: 5G53) were used as search models to perform molecular replacement with the program Phaser. The sequence of mini-Go was modeled using the Swiss-Prot server (46). Structure refinement was performed using phenix.refine (47) and Rosetta refinement (48) from the Phenix suite. Manual model-building and adjustment of the coordinates were performed using the visualization program Coot. Polder maps (49) to verify the presence of retinal were calculated with Phenix using a low-resolution cutoff of 20 Å. For the retinal-only polder omit map, three cross-correlations were calculated between the following maps: (i) calculated Fobs with and without ligand (0.70), (ii) calculated and real Fobs with ligand (0.85), and (iii) calculated and real Fobs without ligand (0.77). These results verified the presence of retinal (fig. S3). In the Fobs-Fcalc (observed and calculated structure factors) map, there is a strong peak density above the 5σ cutoff, and a water molecule was accordingly modeled into this position. The outlier in the Ramachandran plot (R206 in the β3-α2 loop of mini-Go) was likely caused by a crystal contact with ICL3 of rhodopsin in a symmetry-related complex molecule.

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