## 2 Materials and Methods  
**Plasmid construction. ** Plasmid pET-28S-STxB (21-89) was constructed by subcloning a portion (21-89) of the coding sequence from STxB full-length gene, which was a gift from [PERSON] research group (Curie Institute, Paris, France). To amplify this region for recombination with PET-28S vector, we designed a pair of specific primer: 21-F: CACAGAGAACAGATTGTGGAACGCCTG TTGTGTAACTG; 89-R: TGTCGACGAGCTCGAAATTCTCAACGAAAATAA CTTGC. The plasmid was propagated and expressed in _E. Coli_ BL21 (DE3). BL21 was grown aerobically at 37 \({}^{\circ}\)C in Luria broth supplemented with 50 \(\upmu\)g kanamycinin per ml. At an optical density at 600 nm of 0.6, isopropyl-\(\beta\)-d-thioacetamide (IPTG) was added to a final concentration of 0.2 mM. The culture was harvested 18-20 h at 16 \({}^{\circ}\)C following IPTG addition. **Purification of STxB Subunit. ** Bacteria were harvested by tangential flow membrane filtration. The bacteria were pelleted by centrifugation (4000 rpm, 12 min, 25 \({}^{\circ}\)C). The bacteria pellet was resuspended with buffer A (50 mM Tris, pH 8.0; 500 mM NaCl; 5 mM imidazole) to a final volume of 40 mL and was crushed by a high-pressure homogenizer at 4 \({}^{\circ}\)C. Unbroken cells were removed by centrifugation at 18,000 rpm for 12 min at 4 \({}^{\circ}\)C and the supernatant was added to 2 mL Ni\({}^{2+}\)-NTA agarose affinity chromatography. The Ni\({}^{2+}\)-NTA agarose beads were washed 3 times using buffer A for 30 min to remove supernatant, and then 0.1 mg Ubiquitin-like-specific protease 1 (ULP1) prepared with buffer A was added to cut SUMO tag over night at 16 \({}^{\circ}\)C. Purification of B subunit to homogeneity was performed by fast protein liquid chromatography (FPLC) gel filtration. The supernatant was first filtered with 0.22 \(\upmu\)m membrane and further purified by chromatography of the eluted B subunit on a FPLC Superdex 75 Increase 10/300 GL gel filtration column which was equilibrated with buffer B (20 mM Tris, pH 8.0) and run at a flow rate of 0.5 mL/min. The first peak at 280 nm UV were condensed to 10 mg/mL, giving a homogeneous preparation of STxB subunit. The physiological activity of purified STxB subunit was determined by tricine-SDS-PAGE and Western blot analysis using anti-STxB antibody (Sigma, Darmstadt, Germany, 1:2000, Cat# SAB4200774). **Protein Labeling. ** The labeled protein was prepared in reference with [PERSON] et al. (2018) [19] with modifications to improve the expression and purification of STxB. Purified STxB protein were exchanged into the NaHCO\({}_{3}\) buffer containing 100 mM NaCl, 100 mM NaHCO\({}_{3}\) (pH 8.3), 4 mM \(\beta\)-ME using a HiTrap desalting column and concentrated to 10 mg/mL before reaction. Cy3 NHS ester (AAT Bioquest, Sunnyvale, California) were dissolved in DMSO and incubated with the corresponding protein at room temperate for 1 h. The fluorophore was mixed with protein solution in 1:1 molar ratio. The labeling reaction was quenched by the 200 mM Tris (pH 8.2) buffer, and the labeled protein was separated with a HiTrap desalting column into buffer containing 20 mM Tris (pH 8.0). Fluorescence labeling efficiency was measured by Nanodrop 2000 (Thermo Fisher, Waltham, MA, USA). **GUV Formation and Analysis. ** Giant unilamellar vesicles (GUVs) containing the fluorescent lipid \(\beta\)-BODIPY(tm) 500/510 C12-HPC (Thermo Scientific, Waltham, MA, USA) were grown using the thin film hydration method. Briefly, the POPC (Avanti Polar Lipids, Alabaster, AL, USA) and \(\beta\)-BODIPY(tm) 500/510 C12-HPC were dissolved in chloroform and placed in a glass tube. The solvent was dried with nitrogen gas to form a thin phospholipid film and further dried under vacuum for 4 h. After adding saturated sucrose solution, the lipid film was hydrated at 37 \({}^{\circ}\)C under rotary shaking overnight to obtain GUVs. For GUVs containing ganglioside Gb3 (Avanti Polar Lipids, Alabaster, AL, USA), the preparation process was the same as described above, but starting with mixed lipids, POPC and Gb3.