## 3 Results and Discussion
### Model Membrane Thinning upon STxB Binding  
The binding of Shiga toxin to specific glycolipid receptors in the model membrane has been reported to induce membrane deformation locally [15]. This observation was further confirmed in present study using SAXS and SANS. Based on Equation (1), the experimental scattering curves were fitted with the theoretical ones therefore the vesicle bilayer related structural parameters were determined (Figure 3). The fitting residuals of scattering was shown in Figure S3. The scattering data in the small-angle region (\(q\) < 0.15 nm\({}^{-1}\)) was affected by the SUVs size distribution. However, due to the limited measurable minimum \(q\) range, it is hard to follow the scattering of the whole vesicle. Therefore, here we focused more on the internal structure of the SUVs. Figure 3: Optimized theoretical scattering functions on [h-DPPC]/[d-DPPC] = 1/0 system based on polydisperse three-shell model fitting. The SUV were prepared as [Gb3]/[DPPC] = 0.1/1. Measurements were performed at \(T\) = 25 \({}^{\circ}\)C. (**A**) Empty DPPC vesicle; (**B**) Gb3 reconstructed DPPC vesicle; (**C**) Shiga toxin B subunit incubated with Gb3 reconstructed DPPC vesicle. Figure 2: Small-angle scattering curves of the SUV samples of Gb3 and DPPC mixture ([Gb3]/[DPPC] = 0.1/1. Measurements were performed at T = 25 \({}^{\circ}\)C. (**A**) Small-angle neutron scattering curves of [h-DPPC]/[d-DPPC] = 1/0; (**B**) small-angle neutron scattering curves of [h-DPPC]/[d-DPPC] = 0/1; (**C**) small-angle X-ray scattering curves for fully protiated and deuterated DPPC, and fully deuterated DPPC with Gb3. In previous studies, the shell-model was quite popular to be applied for fitting the scattering data. This direct analysis can determine internal structure of micellar or vesicular particles [20; 26; 27]. However, such shell-model fitting analysis neglected the surface structure of micellar or vesicular particles, and was not suitable for present study. Therefore, a more general three shell model was selected (shown in Figure 4). The experimental SAXS and SANS scattering curves were fitted with the integral function (Equations (1) and (3)) at the scattering vector \(q\) ranging from 0.1 to 2 nm\({}^{-1}\) (shown in Figure 3). The value of \(R_{\rm c}\) was fixed at 18 nm as in reference of previous glycosphingolipid vesicle related studies [20; 26]. Overall, the data and fitting curves overlap rather well, except small deviations in the low-\(q\) region (<0.10 nm\({}^{-1}\)), which might be caused by the polydispersity or by attractive interactions between SUVs. The fitted parameters indicated the thinning of the SUV bilayers induced by STxB. Table 2 revealed the effects of Shiga toxin B subunit on the model membrane thickness, with the value of 2.50 \(\pm\) 0.15 nm for STxB-Gb3-DPPC SUV bilayer, which was significantly smaller than that for pure DPPC SUV bilayer (3.33 \(\pm\) 0.05 nm). It should be noted that the membrane thickness for Gb3 ganglioside reconstructed SUV remains stable, with the value of 3.10 \(\pm\) 0.13 nm, which was quite comparable with the pure DPPC SUV bilayer thickness. Thus, the membrane curvature can only be induced in the presence of STxB proteins. The cellular receptor Gb3 itself had little influence on the membrane curvature changes. These results were consistent with studies on another 'rafts' like protein, GPI-APs. [PERSON] et al. reported that there was a small fraction of species with significantly slower diffusion coefficient coexisting with larger oligomers for a GPI-anchored isoform of class II MHC molecules [28]. [PERSON] and his colleagues revealed the size and origin of the slowly diffusing species for GPI-APs using fluorescence photobleaching experiments [29]. Moreover, in [PERSON]'s group, they reported the nanocluster behaviors of GSL GM1 and GM3 in the membrane outer leaflet [30]; and in the inner leaflet of plasma membrane, the nanocluster segregation of Ras isoforms had been discovered [31]. [PERSON] et al.