All image processing was performed in RELION 3.0(51). Beam-induced motion and drift correction were performed using RELION 3.0 own implementation. Contrast Transfer Function (CTF) estimation was carried out using CTFFIND-4.1 (52), with CTF correction carried out on non–dose-weighted micrographs, with further data processing done on dose-weighted micrographs. Around 2000 particles were manually picked and extracted to generate initial 2D classes that were used as templates for automatic particle picking. Approximately 700,000 particles were picked and sorted before 2D classification on ~540,000 particles was carried out. The best 2D classes were chosen to make an initial model, which was then low pass–filtered to 60 Å and used for 3D classification. Several rounds of 3D classification were performed, with the best class out of three being taken forward for the next round of classification. A total of 56,134 particles yielded a refined map at a global resolution of 4.1 Å with C2 symmetry, using a soft mask encompassing the protein and detergent micelle. CTF refinement and Bayesian particle polishing improved the resolution to 3.9 Å, where local resolutions suggest that the core of the protein is 3.7 Å. Tighter masks failed to increase the resolution of the final map. Resolutions were estimated by the gold standard FSC = 0.143 criterion, while local resolutions were valued using RELION. Val495Ala was processed in a similar fashion to the wild type, albeit with motion correction carried out using RELION 3.0 implementation. From ~600,000 auto-picked particles, multiple rounds of 2D and 3D classification led to 227,000 particles being used in the final reconstruction to attain a resolution of 3.7 Å, with C2 symmetry. After CTF refinement and Bayesian particle polishing, the resolution improved to 3.5 Å. A tight mask excluding the micelle was used in postprocessing to improve the resolution further to 3.3 Å, with a local resolution at 3.2 Å. Resolutions were estimated by the gold standard FSC = 0.143 criterion, while local resolutions were valued using RELION. ResMap (40) was used to assess slice through volume local resolution estimates.

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