Figure 4.
Cargo loading mechanisms in Family 2B encapsulins. (A) Disorder plots generated using DISOPRED3 for four different Family 2B cargos.2,74 Yellow boxes in the disorder plots represent the approximate locations of the CLDs. Adapted with open access permission via a creative common license (https://creativecommons.org/licenses/by/4.0/).2 (B) An SDS-PAGE gel of in vivo cargo-loading experiments demonstrating that the CLD is necessary for loading 2-MIBS into the Sg Enc Family 2B encapsulin from S. griseus. Adapted with changes with open access permission via a creative common license.9 (C) Cryo-EM map of the Sg Enc encapsulin loaded with 2-MIBS cargo.17 The Sg Enc shell is colored in blue, the CBDs are green, and the CLD density is yellow. Representative shell-subtracted 2D classes show internal densities corresponding to 2-MIBS. (D) A close-up of the interactions between the conserved interface of the Sg Enc shell and the CLD density of 2-MIBS at the 3-fold symmetry axis. Individual protomers are represented by different background surface shading. The cartoon and stick representations are color coded by amino acid conservation as calculated by ConSurf.73 CLD density is shown in yellow. Adapted with open access permission via a creative common license.9 (E) The amino acid sequence of the 2-MIBS CLD from S. griseus is poorly conserved outside of the consensus motif. Amino acids are color coded according to ConSurf conservation scores.73 Sections of the CLD are labeled and color-coded accordingly. The consensus motif is shown as a sequence logo below the CLD sequence. Adapted with open access permission via a creative common license.9 (F) In vivo cargo-loading experiments using various truncations of the CLD, with each section of the CLD corresponding to the colors in panel (E). The top gel depicts cargo-loaded Sg Enc complexes stained by Coomassie. The bottom gel depicts the observed mNeonGreen fluorescence of cargo-loaded Sg Enc complexes from the same gel. Adapted with open access permission via a creative common license.9 (G) GS-like terpene cyclases have conserved sequence motifs similar to 2-MIBS-like terpene cyclases. Adapted with changes with open access permission via a creative common license.2