Fig. 4: CLD-mediated cargo-loading of Sg Enc.
From: The biosynthesis of the odorant 2-methylisoborneol is compartmentalized inside a protein shell
a Cut-way view of an Sg Enc shell model shown in electrostatic surface representation with extracted cryo-EM CLD densities obtained via asymmetric C1 refinement shown in yellow (left). Zoom-in highlighting CLD density bound to the interior of the shell at the three-fold axis of symmetry (right). b The interior of Sg Enc surrounding the three-fold axis of symmetry shows strong sequence conservation throughout Family 2B encapsulins. Residues are colored based on ConSurf conservation analysis. The three protomers at the three-fold symmetry axis are highlighted by differently colored backgrounds. Conserved hydrophobic residues F430, Y441, L285, and L264 are shown as stick models and are located directly below the observed CLD density (transparent yellow). c AlphaFold model of Sg 2-MIBS highlighting predicted secondary structure elements within the disordered N-terminal CLD. Five CLD elements – CLD_A to CLD_E – are shown in different colors. The structured TC domain of Sg 2-MIBS is shown in surface representation (purple). d Sg 2-MIB CLD sequence colored by ConSurf conservation score. A sequence logo for the conserved motif within CLD_D is shown. Source data are provided as a Source Data file. e SDS-PAGE analysis of purified Sg Enc shells. Deletion of the CLD abolishes cargo loading of Sg 2-MIBS when co-expressed with Sg Enc. Sg Enc_2-MIBS: 2-MIBS-loaded Sg Enc. ΔCLD: truncation mutant missing all of the CLD. This experiment was repeated independently three times. Source data are provided as a Source Data file. f Systematic deletion of the five identified CLD regions – CLD_A to CLD_E (left). A co-expression system of Sg Enc and CLD-fused mNeonGreen (mNeon), employed as a reporter, was used. Native PAGE analysis of purified truncation mutants shown on the right. Coomassie protein stain (right, top) and mNeonGreen fluorescence (right, middle) are shown. Normalized loading percentages based on gel densitometry and fluorescence analysis are shown (right, bottom). Data are shown as mean values, with error bars representing the standard deviation of four independent experiments. Source data are provided as a Source Data file.
