ABSTRACT Polychlorinated biphenyls and organophosphorus pesticides are ubiquitous environmental pollutants. Bioaccumulation of these pollutants in human tissues is associated with cancer and neurotoxicity. They are preferably metabolized by CYP2B subfamily enzymes. Essential to the detoxification of these pollutants are the interactions of CYP2B enzymes with their redox partners, cytochrome P450 oxidoreductase (POR) and cytochrome b5 (cyt b5). However, these interactions in the context of endoplasmic reticulum (ER) membrane remain poorly understood. In particular, the role of cyt b5 in the microsomal P450 system is enigmatic. It has been debated for decades whether cyt b5 affects P450 activity as an electron donor or allosteric effector or both. There is a clear knowledge gap between the important role of cyt b5 in P450 activity and our understanding of its mechanism. Lack of understanding is in part due to the absence of 3D structure of a native P450:b5 complex. It is highly challenging to obtain such a structure by conventional methods like X-ray crystallography or NMR spectroscopy. Single-particle cryo-EM has emerged as the method of choice for structural determination of multi-unit membrane protein complexes where diffracting crystals are not available. We hypothesize that determination of the 3D structure of a native CYP2B4-cyt b5 complex can be achieved using single-particle cryo-EM in conjunction with novel sample preparations. We propose to test the hypothesis and develop a much-needed cryo-EM method in two specific aims. In Aim 1, we will prepare the CYP2B4-cyt b5 complex in novel nanoparticles and then determine its 3D structure by single-particle cryo-EM in Aim 2. Successful completion of the two aims will not only provide critical knowledge for understanding the role of cyt b5 in CYP2B4 activity, but also develop a much-needed methodology for structural analysis of the native complexes of cyt b5 and POR with many other P450 enzymes. Establishment of this methodology and availability of these native complexes will be groundbreaking and provide a roadmap to interrogate the mechanism(s) by which the microsomal P450 system detoxifies environmental pollutants.