Supplementary Figure 12: SsNMR experiments at higher temperatures suggest that T4SScc is largely rigid in the bacterial cell envelope.

a) 1D NCA experiment on BL21dm cell envelope expressing GSLV-T4SScc recorded at -5° C (blue spectrum) and 10° C (red spectrum). No significant change in the spectrum is observed between the two temperatures. This indicates that, at high temperatures, T4SScc is still largely rigid. Taking into consideration that about 25% of the signal in this sample is originating from the N-terminal part of B10, this implies a rigid N-terminal of B10 at this temperature. b) 1D 90° pulse on ¹⁵N performed on the same sample mentioned in a) at 10° C. A prominent sharp lipid peak is visible in the spectrum (~36 ppm), consistent with mobile lipids, and a small broad protein signal is seen at ~120 ppm. This observation further supports the notion of a rigid T4SScc in the cell envelope at high temperatures.