Methanocaldococcus jannaschii, a hyperthermophilic and barophilic methanarchaeon, contains a single gene (MJ0285) encoding a 16.5-kDa polypeptide chain of a small heat-shock protein (sHSP). This sHSP—now called MjsHSP16.5—is upregulated in response to high growth temperature or pressure (1, 2) and functions as a broad substrate ATP-independent holding chaperone that transiently binds and prevents the misfolded proteins from aggregation (3-7). Despite being extensively studied for decades, the molecular mechanism of MjsHSP16.5 is still remained to be elucidated, which primarily required a higher resolution of MjsHSP16.5 structure. In this study, using the single-particle cryo-electron microscopy (cryo-EM) technique which has the capacity in achieving near-atomic resolution of macromolecular structures and preserving the macromolecular shapes in solution, we reconstructed the MjsHSP16.5 24-subunit oligomer to a 2.5-Å resolution. Despite a similar hollow spherical homo-oligomer, the MjsHSP16.5 cryo-EM structure is slightly bigger than its crystal structure and reveals a loosen subunit-subunit interactions. Furthermore, cryo-EM image reconstruction shows additional N-terminal residues which are absent in most of MjsHSP16.5 crystal structures. These residues likely involve the holding chaperone activity and the oligomer stabilization. Using dynamic light scattering (DLS) and negative-staining transmission electron microscopy (TEM), we observed that MjsHSP16.5 oligomer was shrunk upon heating, suggesting a large conformational change in MjsHSP16.5 at elevated temperature. To our knowledge, MjsHSP16.5 is the first sHSP to have the cryo-EM structure archiving a resolution at 2.5 Å.

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