Exploring the complex molecular processes of mitochondrial protein import, this study focuses on the TOM complex and chaperones, employing advanced techniques such as Cryo-EM. The research provides an attempt to high-resolution 3D reconstruction of the yeast chaperone complex Hsp70-Djp1, unveiling crucial insights into mitochondrial precursor protein processing. Additionally, investigations into Human-Tom34 interactions with various solubilization tags reveal distinct specificity patterns. However, the study highlights temporal dynamics in the complex formation of Human-Tom34, Hsp90, and Hsc70StrepII, emphasizing the necessity of a multifaceted approach to comprehend protein-protein interactions. These findings contribute valuable knowledge to mitochondrial biology, potentially informing therapeutic targets for related disorders.