Reconstruction of the Flexible IgM Fc Core Using Atomic Force Microscopy Topography and the AFM-Assembly Pipeline.

Immunoglobulin M (IgM) is the first class of antibody produced in response to pathogens. It also plays an important role in regulating the immune response, including in autoimmunity. IgM pentamers consist of an Fc core composed of Cμ2, Cμ3, and Cμ4 domains, as well as 10 Fab domains composed of Cμ1 and a variable domain. In this study, we use high-resolution atomic force microscopy (AFM) topography to constrain the reconstruction of pentameric IgM Fc core pseudo-atomic model (without Fabs). Reconstruction is performed in two stages using the AFM-Assembly pipeline. First, we fit the known cryo-EM structure of the inner core of the IgM Fc (Cμ3-Cμ4) and the known x-ray structure of Cμ2 beneath the AFM topographs using the DockAFM tool. Second, the two structural units are assembled to generate a complete pentameric IgM Fc core structure. The experimentally constrained reconstruction of the IgM Fc core reveals a divergence in the assembly of the five Cμ2 monomers: the first and last monomers (M1 and M5) are tightly attached to the inner core, while the remaining monomers (M2-M4) are more loosely bound. This reconstruction describes multiple alternative conformations for the attachment of the Cμ2 domains, suggesting that the reconstruction protocol integrates molecular flexibility present in molecules individually deposited on the AFM substrate.