Resolving the polarity of individual actin filaments within bundles by cryo-ET and image processing analysis (A) An x-y slice, 1.4 nm in thickness, through a representative tomogram of a vinculin-induced bundle and its respective surface-rendered view (white frame) displays a detailed view of individual filaments. To favor their random orientation in the solution, preassembled filaments with 0.7 µm median length (where actin:CP was 200:1) were incubated with VBS1-activated vinculin, applied on EM grids, subjected to vitrification, and imaged in their near-native state by cryo-ET (Weber et al., 2019). Scale bar, 100 nm. (B) 2D structural classes were derived from ~30,000 actin segments extracted from 16 tomograms of bundles. (C) Helical reconstruction (Behrmann et al., 2012; He and Scheres, 2017) reveals filament structure and polarity. The helical reconstruction algorithm, as embedded in RELION (He and Scheres, 2017) was employed to generate a 14 Angstrom-resolved structure, in which the filament’s barbed end and pointed end were identified by docking the canonical F-actin structure EMD-6179 (Galkin et al., 2015), with a cross-correlation coefficient of 0.84. (D), Directionality maps of four representative bundles. Using the reconstruction parameters of our structure, actin segments were traced back to their original positions in the 3D tomograms and reconnected to form the original filaments, resolving the directionality of individual actin filaments within the bundles (red and blue, barbed end up and down, respectively). Filaments depicted in black are compromised by a low confidence in the directional assignment (majority score <2/3, see Materials and methods section, Martins et al., 2020). Numbers refer to the bundle’s index in E. (E) Determination of the overall polarity of each bundle as defined by the score (MPs-UPs)/(MPs+UPs). For each segment of a bundle, the degree of uniform polarity (UP) and mixed polarity (MP) describes the orientation of its close neighbors (as detailed in Materials and methods section). Then, for all segments of a bundle, UP and MP values were summed and processed. Bundles with a higher density of uniform-polarity packing (UPs > MPs) have a negative score, while bundles with a higher density of mixed-polarity packing (MPs > UPs) have a positive score. In this analysis, we considered neighbors on either side of the filament.