## 3 Results and Discussion
### Glycolipid organization  
To understand the scales of glycolipid organization at cell surface, deuterated DPPC was used to prepare model vesicles, and small-angle neutron scattering was used to detect GSL microdomains. This methodology has been reasoned by [PERSON] et al. to study the structure and distribution of microdomains in lipid vesicles [PERSON] et al. (2018). After deuteration, the scattering from hydrophobic tail portions of DPPC were minimized. The scattering length density of Gb3 was calculated as 4.78 \(\times\) 10\({}^{11}\) cm\({}^{-2}\); therefore, the changes of SANS curve in Figure 2B in/without the presence of Shiga toxin B subunit were attributed to the Gb3 inhomogeneous distribution in model membranes, and the scattering intensity reflected the shape and size of Gb3 on the surface of SUVs. It has been reported that the glycosphingolipids had a strong tendency to form segregated compositional domains in phospholipid bilayers [PERSON] et al. (2018); [PERSON] et al. (2018); [PERSON] et al. (2018); [PERSON] et al. (2018). Therefore, we hypothesized the Gb3 microdomains form elongated aggregates on the SUV surface, and fitted the scattering curves by the model-independent Guinier law for elongated objects (Equation (3)). The fitted results are shown in Figure 5. The fitting value of Gb3 radius of gyration (\(R_{g}\)) was 5.3 \(\pm\) 0.6 nm (shown in Table S3), which gave evidence that the Gb3 itself could form nanometer-sized clusters on the model membrane. When introducing the Shiga toxin B subunits, the size of Gb3 clusters increased to 7.8 \(\pm\) 0.7 nm (shown in Table S3), indicating that the toxin binding can change the corresponding lipid organization to form larger clusters. These phenomena accounted for the lipid-mediated membrane invaginations. Basically, the lipid domains formation was accompanied with the line tension occurrence between the domains and the rest of the lipid bilayer ([PERSON] et al., 2018). The local change in membrane shape (budding or invagination) can make a balance between the energy needed to bend the bilayer at lipid domains and the penalty paid by having a large contact surface that sets the scale of the bud ([PERSON] et al., 2018). This trade-off process is important for the engagement of viruses and toxins with host transmembrane proteins to enter cells. Lipid-based entry through the induction of membrane microdomains has been reported for SV40 ([PERSON] et al., 2018), Shiga toxin ([PERSON] et al., 2018), and Cholera toxin ([PERSON] et al., 2018), and all these processes can subsequently induce tubular invaginations. In recent reviews, researchers claimed that it was the fluctuation force that might drive toxin clustering on model membranes and in cells ([PERSON] et al., 2018; [PERSON] et al., 2018). The architecture of GSL was selected by convergent evolution to generate the membrane curvature for the clathrin-independent biogenesis of tubular endocytic pits. The schematic representation of STxB subunit induced changes of membrane invagination is shown in Figure 6. With the model membrane of Gb3-DPPC mixed SUVs, the results obtained with SAXS and SANS measurements evidenced that toxin binding could cause changes on membrane curvature, which will further induce compaction of glycolipid into larger domains and the negative curvature on the membrane through toxin-glycolipid clusters to enable changes of membrane thickness. The binding of toxins to cells alone was not sufficient to induce membrane deformation. It was the changes of membrane order, related with the protein clustering capacity, that were responsible for the membrane reorganizations. \begin{table}
\begin{tabular}{c c c} \hline \hline  & **Thickness of Tail (nm)** & **Thickness of Headgroup (nm)** \\ \hline h-DPPC & 3.33 (0.05) & 0.80 (0.08) \\ h-DPPC-Gb3 & 3.10 (0.13) & 0.85 (0.07) \\ h-DPPC-Gb3-STxB & 2.50 (0.15) & 1.13 (0.08) \\ \hline \hline \end{tabular} The data on [h-DPPC]/[d-DPPC] = 1/0 fitted with polydisperse three-shell model. Measurements were performed at T = 25 \({}^{\circ}\)C.  
\end{table}