Supplementary Figure 3: Cryo-EM of the Ll.LtrB group II intron RNP and LtrA-depleted intron.

(a) A representative raw micrograph of frozen-hydrated intron RNP particles generated from the 300 kV Titan Krios microscope. Few typical particles are identified by green square boxes. (b) Computed power spectrum of the micrograph in panel A. (c) Outline of the image processing steps to obtain the 3.8 Å resolution cryo-EM map of the group II intron RNP complex and the 4.5 Å resolution map of the LtrA-depleted intron RNA. Reference-free 2D classification of 608,019 particle images generated ~200 2D class averages of the group II intron RNP comprising good particles free of ice contaminants and other junks. A 3D classification of the selected particles into four classes yielded three classes with strong LtrA protein occupancy that were merged as a total particle dataset of 450,296 particles and one class lacking density for the LtrA protein as a dataset of 102,522 particles. The first round of 3D auto-refinement of the merged dataset yielded a reconstruction at 4.2 Å resolution. A soft mask (shown in light grey semitransparency) around the most rigid portion of the RNP complex was used for further refinement to generate a final 3D reconstruction at 3.8 Å resolution. Local resolution maps of the 3D reconstructions without and with a soft mask during the refinement are shown on the right of the 4.2-Å and 3.8-Å resolution maps, respectively. The volumes are sliced to show the central portion of the local resolution distribution. A refinement of the LtrA-depleted intron dataset yielded a reconstruction at 4.5 Å resolution. The Euler angle distributions of particle images used for the intron RNP and the LtrA-depleted intron RNA reconstructions are shown next to corresponding EM maps. (d) Corrected gold-standard FSC curves of the final 3D reconstructions of RNP or LtrA-depleted intron RNA with or without a soft mask, respectively, after post-processing in RELION 1.3.