We describe the isolation and detailed structural characterization of stable toxic oligomers of α-synuclein that have accumulated during the process of amyloid formation. Our approach has allowed us to identify distinct subgroups of oligomers and to probe their molecular architectures by using cryo-electron microscopy (cryoEM) image reconstruction techniques. Although the oligomers exist in a range of sizes, with different extents and nature of β-sheet content and exposed hydrophobicity, they all possess a hollow cylindrical architecture with similarities to certain types of amyloid fibril, suggesting that the accumulation of at least some forms of amyloid oligomers is likely to be a consequence of very slow rates of rearrangement of their β-sheet structures. Our findings reveal the inherent multiplicity of the process of protein misfolding and the key role the β-sheet geometry acquired in the early stages of the self-assembly process plays in dictating the kinetic stability and the pathological nature of individual oligomeric species.protein misfolding | amyloid aggregation | toxic oligomer | cryoelectron microscopy | neurodegeneration

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A long-standing paradox in the study of T cell antigen recognition is that of the high specificity–low affinity T cell receptor (TCR)–major histocompatibility complex peptide (MHCp) interaction. The existence of multivalent TCRs could resolve this paradox because they can simultaneously improve the avidity observed for monovalent interactions and allow for cooperative effects. We have studied the stoichiometry of the TCR by Blue Native–polyacrylamide gel electrophoresis and found that the TCR exists as a mixture of monovalent (αβγɛδɛζζ) and multivalent complexes with two or more ligand-binding TCRα/β subunits. The coexistence of monovalent and multivalent complexes was confirmed by electron microscopy after label fracture of intact T cells, thus ruling out any possible artifact caused by detergent solubilization. We found that although only the multivalent complexes become phosphorylated at low antigen doses, both multivalent and monovalent TCRs are phosphorylated at higher doses. Thus, the multivalent TCRs could be responsible for sensing low concentrations of antigen, whereas the monovalent TCRs could be responsible for dose-response effects at high concentrations, conditions in which the multivalent TCRs are saturated. Thus, besides resolving TCR stoichiometry, these data can explain how T cells respond to a wide range of MHCp concentrations while maintaining high sensitivity.