Large polysomes are formed by ribosome clusters connected by naked RNA. (a) AFM 3D projections of large HMW polysomes images. To appreciate both ribosome and RNA height levels, the image is shown using different Z-ranges for the color scale (0–0.5 nm gray [background], 0.5–2 nm magenta [RNA], and 2–10 nm yellow [ribosomes]). Thin strands, with height values <2 nm (see yellow arrows), connect clusters of ribosomes ([HMW]ⁿ, 25% of the HMW fraction). (b) 3D projection of HMW polysome formed by two clusters of ribosomes linked by free RNA that appears as a magenta ribbon. Cross-section profiles (solid white line) of RNA connecting adjacent ribosome clusters are shown with corresponding height profile compatible with free RNA. In the right panel, the distribution of naked RNA lengths for 259 [HMW]ⁿ polysomes is shown. (c) Ribosome clusters were considered as single objects, isolated, and classified to unveil the underlying rotational symmetry. (left) The distribution of the clusters forming [HMW]ⁿ polysomes after rotational spectra computation and KerDenSOM classification is shown. Three classes appeared to be the most represented and the MMD was performed. (middle) The top diversities resulting from the MMD test are displayed as a heat map. (right) To highlight any significant similarity in the ribosome organization between these classes ([HMW]ⁿ subclusters) with those found in HMW polysomes (HMW single cluster polysomes) described in Figs. 3 and 4, we compared the corresponding power spectra and ran the MMD over these two populations. Because in this case we are looking for similarities rather than dissimilarities, we plotted the discrepancy values as negative, so the higher the value the higher the similarity. (d) Proposed models of [HMW]ⁿ polysomes (n = 2) formed by assemblies of rounded, rectangular, and triangular clusters of ribosomes (cliques) bridged by free RNA.