Supplementary Figure 4: XL-MS and FRET studies of TcdB holotoxin.

(a) Crosslinking workflow used to unambiguously identify DSSO crosslinks from 3 replicates (Rep1–3) of cross-linked TcdB holotoxin. (b) MSⁿ identification of a DSSO inter-linked peptide from TcdB: K1117–K2249. The cross-linked peptide α–β⁴⁺ (m/z 510.991⁴⁺) was detected during MS¹. Next, it was selected for MS² where it was fragmented into two characteristic fragment ion pairs, that is α_A/β_T (m/z 308.67²⁺/704.31²⁺) and α_T/β_A (m/z 324.65²⁺/688.32²⁺). Finally, these peaks were selected for MS³ analysis. α_A (m/z 308.67²⁺) identified the sequence DK_AATK, in which the lysine residue at position 2 was modified with an alkene moiety. β_T (m/z 704.31²⁺) identified the sequence as YYFDEK_TGIMR, in which the lysine residue at position 6 was modified with unsaturated thiol moiety. (c) Dynamic light scattering showed a monodispersed peak for TcdB holotoxin at neutral pH. (d) The distribution plot of identified linkages versus their spatial distances measured in the structure of TcdB holotoxin. (e) 5D has no effect on the fluorescence emission of the dye-labeled VHHs and the TcdB–B39–7F complex based on an ensemble FRET study. TcdB holotoxin was incubated with equimolar ratio of the Alexa-555-labeled B39, the Alexa-647-labeled 7F, or both VHHs, with or without 5D at pH 7 or pH 5. Fluorescence emissions upon excitation by 540 nm were measured. Data are the average of duplicated experiments. (f) Fluorescence intensity over time for single molecules of TcdB in complex with dye-labeled VHHs. Alexa 555, donor-labeled B39 (magenta). Alexa-647, acceptor-labeled F7 (cyan). Data was recorded at a frame rate of 10 Hz. Only molecules showing single step photobleaching to baseline for both dyes were included in the analysis. From the magnitude of the anticorrelated bleaching event, we obtained the γ-correction factor for each molecule that allows us to report the absolute FRET efficiencies. FRET was calculated for each frame until acceptor photobleaching. (g) Histogram of average FRET efficiency for each molecule at either pH 5.0 (filled circles, n = 498) or pH 7.0 (open circles, n = 594). This reports a single value for each molecule, which averages out any conformational dynamics. The mean FRET efficiency was unchanged by changing the time binning of the data. (h) Mean smFRET efficiency for TcdB in complex with dye-labeled VHHs at intermediate pH. FRET was calculated from each 100 ms frame of the movie until acceptor photobleaching, compiled into histograms and fit to a Gaussian function to obtain the FRET efficiencies. Error values represent the standard deviation from replicate measurements.