Diagram of the molecular subsystems shown to be active in the Cytomim cell‐free system. Diagram of the molecular subsystems shown to be active in the Cytomim cell‐free system. Glutamate (GLU) is used as a robust energy source in a natural chemical environment to produce reducing equivalents, primarily in the form of NADH, through the TCA cycle. NADH fuels oxidative phosphorylation in which oxygen serves as the final electron acceptor. Membrane‐dependent respiration provides a stable supply of ATP, which is coupled to combined transcription and translation followed by protein folding. To prove the existence of oxidative phosphorylation in the Cytomim cell‐free system, inhibitors were used to inactivate the electron transport chain (ETC), dissipate the proton motive force (PMF), and inactivate the F1FO‐ATPase enzyme complex (each is denoted with an *) (αKG, alpha‐ketoglutarate; SUC, succinate; MAL, malate; PYR, pyruvate; OAC, acetate; OAA, oxaloacetate; ASP, aspartic acid; Pi, inorganic phosphate; TFs, translation factors; aa‐tRNAs, aminoacylated‐tRNAs). Michael C Jewett et al. Mol Syst Biol 2008;4:220 © as stated in the article, figure or figure legend