Fig. 2. Near atomic resolution reconstruction and characterization of the PDHc core.

a Icosahedrally-averaged cryo-EM reconstruction of PDHc core. Densities corresponding to PDHc higher-order binders, visible in raw micrographs (scale bar represents 10 nm), as well as external PDHc densities of the metabolon, visible in 2D classes, are averaged to retrieve the high-resolution core structure. Each E2 monomer is colored uniquely. b Comparison of improvement in resolution, compared to previous data²¹. CoA molecule is not resolved but is here computationally placed and refined within the binding site as described in the Methods section. Sidechains in the CoA binding pocket of the active site are distinguishable. Helical pitch (arrowhead) allows now the unambiguous placement of the backbone. c Energetic calculations within the E2 core structure from n = 50 HADDOCK models. The intra-trimeric building block with an extensive buried surface area (BSA) is stabilized by electrostatics (ES) and van der Waals (vdW) energies, whereas desolvation energies (DS) have a minor contribution. In the dimeric inter-trimeric interaction, DS energy plays a major role, whereas vdW and ES energies have decreasing contributions. The box minima represent the 25th percentile, the box maxima the 75th percentile, the Notch indicated the data’s median, whiskers extend to the minimum and maximum value inside of a 1.5 interquartile range. All data points are overlayed as a beeswarm plot. d Potential Arginine cluster in the intra-trimeric interface. Arg384 of each subunit are in close contact, thereby contributing to the electrostatic binding energy. Contour levels applied for the cryo-EM maps are as follows: a 0.05, b 0.041, c 0.03 (new map) and 0.09 (old map).