Fig. 1: SidJ E565A/CaM and SdeA form a stable reaction intermediate complex.
From: Structural basis for protein glutamylation by the Legionella pseudokinase SidJ
A Size-exclusion chromatography (SEC) profiles of SidJ/CaM and SdeA in the presence of ATP. The highlighted fraction in the SidJ/CaM E565A + SdeA sample is shown on SDS-PAGE (right). B SEC profiles of SidJ/CaM E565A + SdeA in the presence of various cofactors. C Schematic representation of the reaction scheme of SidJ-mediate glutamylation of SdeA highlighting the trapped adenylylated reaction intermediate between SidJ/CaM and SdeA. D Time course experiment measuring the incorporation of [14C]-Glu into SdeA catalyzed by SidJ using SidJ WT, SidJ E565A. Samples were separated by SDS-PAGE and visualized by Coomassie stain and autoradiography (Top). The experiment was done in triplicates and [14C]-bands were quantified and plotted (Bottom). Purple corresponds to WT, blue corresponds to the E565A mutant. Individual measurements are shown. E Flow chart of the sample preparation process of SidJ E565A/CaM + SdeA for Cryo-EM. From left to right, samples are incubated and then cross-linked via GraFix method. Fractions are analyzed via SDS-PAGE and further purified via SEC.
