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Bacterial methane oxidation using the enzyme particulate methane monooxygenase (pMMO) contributes to the removal of environmental methane, a potent greenhouse gas. Crystal structures determined using inactive, detergent-solubilized pMMO lack several conserved regions neighboring the proposed active site. We show that reconstituting pMMO in nanodiscs with lipids extracted from the native organism restores methane oxidation activity. Multiple nanodisc-embedded pMMO structures determined by cryo–electron microscopy to 2.14- to 2.46-angstrom resolution reveal the structure of pMMO in a lipid environment. The resulting model includes stabilizing lipids, regions of the PmoA and PmoC subunits not observed in prior structures, and a previously undetected copper-binding site in the PmoC subunit with an adjacent hydrophobic cavity. These structures provide a revised framework for understanding and engineering pMMO function. Description Getting the environment just right Particulate methane monooxygenase (pMMO) is a key enzyme for the metabolism of methane by aerobic, methanotropic bacteria. There is considerable interest in understanding this reaction, both as a fundamental biogeochemical transformation and for potential biotechnological applications, but progress has been hampered by inactivation of this membrane-bound metalloenzyme upon purification. Koo et al. reconstituted natively produced pMMO in lipid nanodisks and found that restoration of the bilayer environment also restored activity of the enzyme. A series of cryo–electron microscopy structures revealed previously missing regions of the enzyme, including conserved residues that coordinate a metal ion near the inner pore of the trimeric complex in a site not seen in previous structures. Lipids play an important role in stabilizing various parts of the structure, and the recovery of activity in these samples is consistent with the newly observed metal-binding site being involved in methane oxidation. —MAF Reconstitution of a copper enzyme in a lipid bilayer restores methane oxidation and gives insight into active site structure.