## 1 Introduction  
Membranes of eukaryotic cell are of well-regulated environments that can provide a dynamic platform for various cellular processes. They are also characterized by high membrane heterogeneity including discrete membrane domains formed through the protein and lipid interactions [1, 2]. It is the vesicular trafficking that regulate and maintain the constant of protein and lipid contents and further keep the heterogeneous environment on eukaryotic cell membrane [3, 4]. Previous studies showed that such membrane heterogeneity is important for cell viability and some pathologies [5, 6, 7]. Therefore, it is of great importance to understand the functional properties of membrane domains in eukaryotic cell.  
Among a variety of hypothesis about the structure and function of membrane domains, one important example is GSL-enriched microdomains on membrane which may facilitate the clathrin-independent endocytic process [8,9]. Such clathrin-independent endocytosis pathways do not involve with coated pits, and account for the cargo segregation during endocytosis [10,11]. The mechanisms for cargo segregation can be broadly categorized into active or passive processes [11]. A good example for active process that depends on ATP hydrolysis is the active segregation of GPI-anchored proteins (GPI-APs), with their organizations on cell surface presenting as monomers and small fraction (20-40%) of nanoclusters (<5 nm) [12]. The passive processes are not energy-dependent and the cargo segregation depends on specific intermolecular cargo interactions [11]. Lipid structure, height of the membrane bilayer and membrane curvature may contribute to such passive clustering of membrane components [11,13,14].  
Intoxication of cells by Shiga toxin produced by Shigella dysenteriae requires its binding to the GSL receptor Gb3, a raft-favoring lipid. In this case, the pentavalent Shiga toxin B-subunit (STxB) can specifically bind to 15 Gb3 molecules and induce narrow tubular vesicles. The compaction of Gb3 lipids under Shiga toxin binding, which favor the negative membrane curvature formation, was thought to be the driving force [15]. A recent study on Shiga toxin argued that the toxin clustering was membrane mediated [16], and the clustering was strictly correlated with capacity of toxins to suppress membrane fluctuations. The membrane thickness was assumed to be closely related with the strength of such fluctuation-induced forces [17].  
In the current work, a simple purification scheme for the isolation of STxB was set up. The purified B subunit was found to exist as a pentamer in native state, and its immunologic and biologic activities were properly characterized. Then, the surface organization of GSL with and without GSL-dependent toxin on model membrane, Gb3-DPPC mixed SUVs was determined by SR-SAXS and SANS. The invagination of GSL-DPPC membrane induced by toxin B-subunit was quantified for the first time, indicating a possibility of membrane fluctuation. The observed experimental clustering processes operating in correlation with biological membranes provide evidences to the thermal Casimir force [18] hypothesis, which is expected to apply to a wide range of membrane biological processes.