Three microliters of purified NRTN-GFRα2- RET complex at a concentration of ~1.6 μM was incubated for 10 s on glow-discharged holey gold grids (UltrAuFoil R1.2/1.3) in a Vitrobot MkIV (3-s blot, 4°C, 100% humidity). Images were collected on a 300-kV FEI Titan Krios electron microscope using a slit width of 20 eV on a GIF Quantum energy filter. A Gatan K2 Summit detector was used in counting mode at a magnification of 130,000 (yielding a pixel size of 1.05 Å) and a dose rate of ~5.2 electrons per pixel per second. Exposures of 8 s (yielding a total dose of 38 eÅ−2) were dose-fractionated into 20 movie frames that were stacked into a single MRC stack using newstack. A total of 2242 images were collected using E Pluribus Unum (EPU) automatic data collection across two independent sessions with defocus values ranging from 0.4 to 4.5 μm and a stage tilt of 40°.

Collected micrographs were corrected for local-frame movement and dose-filtered using UcsfDfCorr (35). The contrast transfer function parameters on a per-particle basis were estimated using GCTF-0.5 (36), and RELION-2.1.b1 was used for all other image processing steps. (37). The two datasets were processed independently up to 3D refinement, where the particles were then joined within RELION. Particles (340,661) were picked using reference-based picking, and bad particles were removed by extensive reference-free 2D class averaging, resulting in 186,903 particles for 3D auto-refinement. This yielded a consensus reconstruction with a nominal resolution of 7.0 Å. Fine-angular 3D classification was used to further identify flexibility in the complex yielding four classes with resolutions ranging from 7.4 to 9.1 Å. Since the complex is a dimer, the reconstruction of the monomer was improved by symmetry expansion. Particles were rotated around the C2 axis, and one copy of the dimer was signal subtracted. The remaining copy was aligned to obtain a 5.7-Å resolution consensus monomer reconstruction.

Reported resolutions are based on gold-standard refinement applying the 0.143 criterion on the FSC (Fourier Shell Correlation) between reconstructed half-maps. The FSC was corrected for the effects of a soft mask using high-resolution noise substitution (38). All 3D refinements used a 60-Å low-pass–filtered initial model, the first of which was a reconstruction calculated ab initio using RELION. Before visualization, all density maps were corrected for the modulation transfer function of the detector and then sharpened by applying a negative B-factor that was estimated using automated procedures (39).

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