Supplementary Figure 1: Crystal structure of the TcdB–5D–E3–7F complex and the representative electron density maps.

(a) A 2Fo-Fc electron density map for the CROPs (residues G1835–E2367) contoured at 1.0 σ. (b–d) Representative 2Fo-Fc electron density maps for the newly identified SR (residues G1815–S1834) and two other regions in the CROPs (residues G1898–T1927 and G2345–E2367) contoured at 1.0 σ. The bulky residue Tyr was used as one of the markers to confirm the sequence register of the structural model. (e) An anomalous-difference electron density map generated from a crystal of the TcdB–5D–E3–7F complex soaked in tantalum bromide. The map was calculated using a 5.44 Å resolution data collected at λ = 1.2524 Å and phases derived from the final structure model. We found 10 tantalum bromide molecules in one asymmetric unit, which all bind to negatively charged glutamate or aspartate residues and thus confirmed the amino acid register of the complex. One representative tantalum bromide peak was shown in the inset. (f) Overall structure of the TcdB–5D–E3–7F complex. The color scheme for the TcdB domains are the same as that shown in Fig. 1. The three VHHs are colored purple (5D), brown (E3), and green (7F). (g) The structure of TcdB holotoxin is fitted into the negative stain EM map of TcdA that is displayed as a grey surface model (map was generously provided by Dr. Lacy D.B.) (Chumbler, N.M. et al., Nat Microbiol. 1, 15002, 2016). The CROPs of TcdA interacts with the DRBD and adopts a closed conformation.