Annealing is an ideal approach to synchronizing soluble proteins into their minimum-energy states via tandem heating and cooling treatments. Like soluble proteins, many membrane proteins also suffer intrinsic structural flexibility, the major obstacle to high-resolution structural determination. How to apply annealing onto membrane proteins remains unexplored. Here, we utilized the translocase of the outer mitochondrial membrane (TOM) as the model and investigated the ideal annealing conditions for membrane proteins. After structural determination via cryo-electron microscopy, we indicated that fast cooling the heated TOM complex to 0 °C can significantly improve the local resolution compared with the unannealed one. Structural analyses showed that annealing renders the TOM complex into a new conformation with its Tom7 α1 helix from a reclining position on the membrane surface to a lying orientation, accompanied by the loop between β6 and β7 in Tom40, flipping outward from the Tom40 β-barrel, ideal for preprotein translocation. In all, our results demonstrate the role of annealing in synchronizing membrane proteins and unveil unidentified conformations of the TOM complex.