AgsA (aggregation-suppressing protein) is an ATP-independent molecular chaperone machine belonging to the family of small heat shock proteins (sHSP), and it can prevent the aggregation of non-natural proteins. However, the substrate-binding site of AgsA and the functional unit that captures and binds the substrate remain unknown. In this study, different N-terminal and C-terminal deletion mutants of AgsA were constructed and their effects on AgsA oligomer assembly and chaperone activity were investigated. We found that the IXI motif at the C-terminus and the alpha-helix at the N-terminus affected the oligomerization and molecular chaperone activity of AgsA. In this work, we obtained a 6.8 angstrom resolution structure of AgsA using Electron cryo-microscopy (cryo-EM), and found that the functional form of AgsA was an 18-mer with D3 symmetry. Through amino acid mutations, disulfide bonds were introduced into two oligomeric interfaces, namely dimeric interface and non-partner interface. Under oxidation and reduction conditions, the chaperone activity of the disulfide-bonded AgsA did not change significantly, indicating that AgsA would not dissociate to achieve chaperone activity. Therefore, we concluded that the oligomer, especially 18-mer, was the primary functional unit. (C) 2020 Elsevier Inc. All rights reserved.