Supplementary Figure 3: Designed substrates are stably bound to needle complexes.
From: Structure of a pathogenic type 3 secretion system in action
(a) Needle complexes (NCs) were purified from Salmonella strain SB905 as well as from strains expressing SptP1-GFP or SptP3-GFP. Substrates co-purified with NCs in CsCl gradient fractions. Top panel: Coomassie stained gels (4-20% SDS-polyacrylamide) of individual CsCl fractions. Lower panels: Western blotting of corresponding samples (rabbit anti-NC; rabbit anti-SptP, and mouse anti-FLAG). (b) Pooled CsCl fractions containing NCs (WT) or NCs and substrates (SptP1-GFP, SptP3-GFP) were subsequently separated on sucrose gradients. Western blotting results showed that designed substrates (SptP1-GFP, SptP3-GFP) are stably associated into NCs. Isolation of WT NCs showed no co-migration of natural substrates. (c) Peak fractions of sucrose gradients of w.t., and substrate-bound complexes (+pSptP1-GFP and +pSptP3-GFP) are negatively stained (2% PTA) for analysis by transmission electron microscopy: In total 27% of NCs containing the substrate SptP3-GFP displayed a clearly visible needle-tip density (ntotal= 298; ntip-density = 83). In contrast, all (100%) WT (substrate-free) NCs (ntotal= 287) were free of a visible density at the needle tip.
