Extended Data Fig. 2: Cryogenic electron microscopy (Cryo-EM) data collection and analysis.
From: InsP6 binding to PIKK kinases revealed by the cryo-EM structure of an SMG1–SMG8–SMG9 complex
Cryogenic electron microscopy (Cryo-EM) data collection and analysis. (a) A representative cryo-EM micrograph collected on an FEI Titan Krios microscope, operated at 300 kV and equipped with a K2 Summit camera. (b) Representative reference-free 2D class averages. (c) Angular distribution of the particles used for the final round of refinement. (d) Local-resolution analysis of the SMG1c. Map shows the variation in local resolution, as estimated by RELION. (e) Local-resolution analysis of the SMG1c (including the C-terminal region of SMG8). Map shows the variation in local resolution, as estimated by cryoSPARC. (f) 3D FSC and preferred orientation analysis of the dataset with the red line representing the estimated global FSC of 3.45 Å ± 1 SD (green dashed lines). A sphericity of 0.943 indicates a mostly isotropic map without preferred orientation bias. (g) Model vs. map FSC for the final PHENIX real-space refined model. (h) The model was probed for over-fitting by randomly perturbing the atoms by 0.5 Å and refining against the first of the two independent half-maps (work half-map, red). The resulting refined model was then used to calculate a model-map FSC against the second half-map (test half-map, green), which was not used for refinement. FSC-work and FSC-test curves show excellent agreement over the entire resolution range, validating the entire structure against over-fitting.
