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Knowing the mechanism of how a polypeptide chain folds into its functional conformation is important for protein engineering and protein drug designs. To date, more than 1% of the Protein Data Bank (PDB) entries have been identified to contain topologically knotted elements, which present a puzzle to challenge biophysicists. In order to gain more insights into the folding mechanisms of these knotted proteins, we use a trefoil knotted MJ0366 from M. jannaschii as a model system. MJ0366 is the smallest knotted protein that has been identified to date and belongs to the ribbon-helix-helix (RHH) superfamily of DNA binding proteins. The crystal structure of MJ0366 (PDB code: 2EFV) forms a homodimer with a typical α/β knotted motif as seen in many knotted proteins. The dimerisation state is confirmed by SEC-MALS in aqueous conditions. Systematic folding analyses of MJ0366 are carried out using circular dichroism, fluorescence, SAXS and NMR. On the basis of these biophysical results, we hope to establish the folding pathway of this small knotted protein and therefore help shed light on the understanding of how knotted proteins fold themselves.