We report an approach for determining the structure of macromolecular assemblies by the combined application of cryo-electron microscopy (cryo-EM) and site-directed spin labeling electron paramagnetic resonance spectroscopy (EPR). This approach is illustrated for Hsp16.5, a small heat shock protein that prevents the aggregation of nonnative proteins. The structure of Hsp16.5 has been previously studied by both cryo-EM and X-ray crystallography. The crystal structure revealed a roughly spherical protein shell with dodecameric symmetry; however, residues 1-32 were found to be disordered. The cryo-EM reconstruction at 13 A resolution appeared similar to the crystal structure but with additional internal density corresponding to the N-terminal regions of the 24 subunits. In this study, a systematic application of site-directed spin labeling and EPR spectroscopy was carried out. By combining the EPR constraints from spin label accessibilities and proximities with the cryo-EM density, we obtained an atomic model for a portion of the Hsp16.5 N-terminal region in the context of the oligomeric complex.