Fig. 4. Connecting filamentation and function of AtP5CS.
a, Negative-staining EM micrographs of AtP5CS2 GK domain incubated with different substrates. Regions in red rectangles are zoomed in on the right of the original micrographs. Scale bars, 200 nm for original micrographs and 50 nm for zoomed-in micrographs. b, Statistical analysis of AtP5CS2 filament length under different conditions. Violin plots show the medians (solid lines) and quartiles (dashed lines). Two-sided Mann–Whitney test was used. c, Negative-staining results of AtP5CS2-WT and AtP5CS2-F80A. Scale bars, 100 nm. d, GPR activity of AtP5CS2-WT and AtP5CS2-F80A, measured by the change of absorbance at 340 nm (A340). Error bars show the upper and lower bounds of data (n = 3 technical repeats). e, GK activity of AtP5CS2-WT and AtP5CS2-F80A, measured by phosphate generation. Error bars show the upper and lower bounds of data (n = 3 technical repeats). f, Model of AtP5CS2 filament dynamics. The blue rounded rectangles represent GK tetramers, and the orange sticks represent hooks. Two different states of hooks are shown in this figure: the open one (APO and products released states) and the locked one (substrates binding and catalysing states). g, Possible mechanisms of how a filament contributes to substrates channelling. Three GK tetramers (grey) and one GPR dimer (light pink or light blue) are shown here. GPR dimer can rotate up and down around the central GK tetramer, and one farthest position of GPR dimer is shown here. The activity centre of AtP5CS2 GPR is inferred from DmP5CS model (PDB: 7WXI), the key amino acid C543 for catalysing is shown. The red arrows show two possible G5P transfer paths.