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Cryo–electron microscopy (cryo–EM) is widely used to determine macromolecular structures at atomic resolution. The theoretical size lower limit of particles for cryo–EM analysis is 38 kDa, limited by factors such as contrast and particle alignment accuracy. To date, no cryo-EM structures have been reported for proteins near this size limit. This study presents cryo-EM structures of two protein-ligand complexes around 40 kDa. The structure of maltose-binding protein (43 kDa) was determined at 2.32 Å resolution, clearly revealing the bound maltose and water molecules. Additionally, the kinase domain of human PLK1 (37 kDa), slightly below the theoretical limit, was determined at 3.04 Å resolution, allowing the identification of the bound ligand, onvansertib. These findings demonstrate that cryo–EM can be effectively employed for structure determination and structure-based drug screening of small proteins or domains.