Fig. 2: Architecture and contact map of the VgrG-Rhs1-EagR complex.
From: Mounting, structure and autocleavage of a type VI secretion-associated Rhs polymorphic toxin
a Schematic representations of Photorhabdus laumondii VgrG and Rhs1 proteins. The protein domains are indicated, as well as their boundaries. b–d Pull-down assays. Total cell extracts (T) from E. coli BL21(DE3) cells producing Strep-tagged EagR (STEagR), FLAG-tagged Rhs1 (Rhs1FL) with His6-tagged VgrG (HVgrG) truncated variants (b) or His6-tagged VgrG (HVgrG), Strep-tagged EagR (STEagR) with FLAG-tagged Rhs1 (Rhs1FL) truncated variants (c and d) were subjected to purification on streptactin-agarose beads. Strep-tagged and co-precipitated proteins were eluted with desthiobiotin (IP). Total and IP fractions were analyzed by SDS-PAGE and co-purified proteins were stained by Coomassie blue (upper panel) or immunodetected using anti-His, anti-Strep, and anti-FLAG antibodies (lower panels). Molecular weight markers (Mw, in kDa) are indicated on the left. Pull-down assays have been performed at least in triplicate, and a representative experiment is shown. e Schematic representation of intermolecular and intramolecular contacts between the EagR, Rhs1, and VgrG proteins identified by cross-link mass spectrometry. For clarity reasons, only the contacts that have minimal distance of 50 amino acids are shown. All cross-linked peptides are listed in Supplementary Table 1, and a map with all the contacts is shown as Supplementary Fig. 5. f Schematic model of the Photorhabdus laumondii VgrG (shades of orange)-EagR (blue)-Rhs1 (shades of red) complex architecture based on pull-down and cross-link mass spectrometry experiments.
