Extended Data Fig. 3: Cryo-EM processing and model building.
A representative section of one of 10,224 micrographs is shown. Graphene oxide (GO) edges were removed in cycles of initial 2D and one 3D classification. The negative stain reconstruction (see Extended Data Fig. 2) was used as initial model. 3D classes were centered in the box by applying a coordinate transformation to the alignment parameters, and unbinned particles were re-extracted with recentering. Particles were filtered for high-resolution features in cycles of 3D refinement, classification, Bayesian polishing, and CTF refinement as indicated. b. Postprocessed map (B-sharpened with -51.1 Å2, contoured at 4.9 σ) with local resolution. c. angular distribution. d. Fourier Shell Correlation (FSC). e. Multibody refinement improved map quality, but not the overall resolution. Considerable improvement of map quality was achieved by filtering with LocSpiral50. The model for the core and TRRAP was built into the LocSpiral filtered maps of the multibody refinement. The interface between these regions was built using the full map and used for model refinement. Refinement against the postprocessed map (b) resulted in the same model, with virtually identical statistics and an all-atom r.m.s.d. of 0.400 Å. Maps are contoured at (regular/LocSpiral): Core 11.2 σ/9.2 σ, TRRAP 7.9 σ/9.0 σ, full 6.9 σ. f. Map vs. model FSC using the postprocessed map shown in b. g. The refined map shows well defined secondary structure elements and side chains (contoured at 9.0 σ). h. Model-sequence coverage. Sequences of all subunits are indicated as horizontal lines (black) and modeled regions as overlaying boxes (orange: visualized by cryo-EM; blue: visualized only by negative stain; translucent: regions with unclear register assignment (unknown, UNKs)). i, j. The LocSpiral filtered multibody map of the core reveals additional density corresponding to the poorly ordered TAF6L HEAT domain, and to SUPT3H in the cleft between the core and TRRAP module (both contoured at 5.9 σ).
