## 3 Results and Discussion
### Contrast Dependence of the Scattering Curves  
To study the lateral segregation of glycolipids on model membrane, the varied inverse contrasts of [Gb3]/[DPPC] = 0.1/1 mixture were measured using SANS, where the molar ratios of [h-DPPC]/[d-DPPC] were 1/0 (shown in Figure 2A) and 0/1 (shown in Figure 2B), respectively. Figure 2C shows the SAXS curves of vesicles, which are prepared in the same way as those used for SANS measurements. All X-ray scattering curves measured at the same temperature agreed well with each other, demonstrating the same contrast profiles for the measured SUVs. In considering that the head and tail portions for DPPC molecules (either h-DPPC and d-DPPC) possessed the same X-ray scattering length densities, the scattering profile of SUVs should be independent on deuteration of DPPC. More changes of SANS scattering curves have been observed in Figure 2B. The average scattering length density of the hydrophilic head portions of h-DPPC and d-DPPC molecules for neutron were identical (1.10 \(\times\) 10\({}^{10}\) cm\({}^{-2}\)). However, with the use of deuterated phospholipid, the scattering length density of the hydrophobic tail portions of DPPC molecules for neutron had high contrast (shown in Table 1) [24; 25]. To minimize the scattering from hydrophobic tail portions of DPPC and hence mask the scattering from phospholipid, the molar ratio of [h-DPPC]/[d-DPPC] = 1/0 mixture was chosen. 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 without the presence of Shiga toxin B subunit were attributed to the Gb3 inhomogeneous distribution in model membranes. The scattering length densities calculated with these parameters across the membrane are shown in Table 1. The scattering of Figure 2 illustrated with error bar was shown in Figure S2.  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline \begin{tabular}{c} SLD \\ (10\({}^{-11}\) cm\({}^{-2}\)) \\ \end{tabular} & \begin{tabular}{c} **Isner Part** \\ **[h-DPPC/d-DPPC] = 1/0** \\ \end{tabular} & \begin{tabular}{c} **Headgroup** \\ **[h-DPPC/d-DPPC] = 1/0** \\ \end{tabular} & \begin{tabular}{c} **Inner Part** \\ **[h-DPPC/d-DPPC] = 1/0** \\ \end{tabular} & \begin{tabular}{c} **Headgroup** \\ **[h-DPPC/d-DPPC] = 0/1** \\ \end{tabular} & \
\begin{tabular}{c} **Gb3** \\ **[h-DPPC/d-DPPC] = 0/1** \\ \end{tabular} &
\begin{tabular}{c} **D\({}_{2}\)O** \\ \end{tabular} \\ \hline Without Gb3 & 2.3 & 11.0 & 66.2 & 11.0 & 4.8 & 63.7 \\ With Gb3 & 2.5 & 11.2 & 58.2 & 11.2 & \\ \hline \hline \end{tabular}
\end{table}
Table 1: The scattering length density used to determine contrast distribution profiles of the SUV bilayer structures for neutron scattering (\(\times\)10\({}^{-11}\) cm\({}^{-2}\)). The SUV samples of Gb3 and DPPC mixture was prepared with [Gb3]/[DPPC] = 0.1/1. The SLD of hydrophilic head portions of h-DPPC and d-DPPC were identical, the high contrast comes from the hydrophobic tail portions of DPPC molecules.  
Figure 1: Novel pentamer Shiga toxin B subunit reconstitution method. The Shiga toxin B subunit purified with novel strategy can bind with Gb3 glycosphingolipids in GUVs. GUVs were composed of DPPC (spiked with 1 mol% BodipyFL-C5-HPC, Green), containing Gb3 ganglioside. [Gb3]/[DPPC] = 0.1/1; STxB (Cy3, Red) was incubated with GUVs at 37 \({}^{\circ}\)C.