A detailed, three-dimensional spatial model of methanogenesis proteins would be useful in these efforts. Metabolic reactions that are linked by multienzyme complexes have clear advantages over reactions that are catalyzed by individual, unlinked enzymes. Complexes channel substrates to prevent diffusion of intermediates into bulk cytoplasm, effectively increasing the relative local concentration of reactants in subsequent pathway steps, speeding the overall rate of production of the final product, and preventing diffusion of toxic intermediates that can damage cell constituents. Complexes can also provide a means of co-regulating pathway enzymes or ensuring proper enzyme dosage. Methanogens obtain up to 1 mole ATP per mole substrate consumed and live near the thermodynamic lower limit of life. Substrate channeling via multienzyme complexes would provide a kinetic advantage by ensuring Scoparone maximal efficiency for converting substrate to ATP generation. We used in vivo crosslinking, tandem affinity purification, and peptide mass spectrometry to look for complex formation among methanogenesis enzymes. XL-MS is a reliable technique for identifying protein:protein interactions by identifying crosslinked partners which elute together after affinity column purification. A recent effort in Saccharomyces cerevisiae has successfully demonstrated the ability to use XL-MS to reproduce 30 years of protein:protein interaction data and to predict new interactions which were subsequently verified genetically. Though commonly applied to the study of cell signaling networks, we surmised that XL-MS is a valuable technique for identifying protein:protein interactions between methanogenesis enzymes and electron transfer proteins in the methanogen, Methanosarcina acetivorans. The majority of cultivated methanogen strains are restricted to using formate or CO2 as the sole carbon source, and these methanogens use the hydrogenotrophic methanogenic pathway, which relies on Ginsenoside-Re reducing equivalents from hydrogen gas to reduce formate or CO2 to methane.