Supplementary Figure 4: Visibility of substrate domains from SptP3-GFP bound to needle complexes.
From: Structure of a pathogenic type 3 secretion system in action
(a) Flexibility of emerging substrate at the tip of needle complexes. Collection of class averages of immuno-purified, substrate-trapped (SptP3-GFP) needle complexes. Class averages were calculated from negatively stained (2% (w/v) PTA) needle complexes. The different position of the needle-tip density comprising the emerging substrate (SptP3-GFP) and its diffuse character indicated a high flexibility and/or folding state of this domain. (b) Localization of the C-terminal substrate domain in substrate-trapped injectisomes Immuno-labeling (anti-GFP-gold) of substrate-free and substrate-trapped (SptP3-GFP) needle complexes on the electron microscopy grid. Highly specific labels close to the basal site of needle complexes could only be found in the substrate-trapped sample (2% (w/v/) PTA). (c) Sketch of a selection of various possible substrate-trapped injectisomes. The substrate might be trapped at various vertical positions and the C-terminally fused GFP in SptP3-GFP can be present at various horizontal positions relative to the needle complex (cbd = chaperone binding domain; N-term = N-terminal region; GFP = green fluorescent protein) (d) GFP (encircled with orange dots) is visible in the difference volume by lowering the threshold for the volume display (34.3 to 26.3).
