5,10-Methenyltetrahydrofolate synthetase (MTHFS) is an essential enzyme of the one-carbon metabolic network, which supplies vital components for the growth and proliferation of cells. Human MTHFS (hMTHFS) has been touted as a promising target for cancer chemotherapy, since inhibition of MTHFS by a folate analog, 5-formylTHHF, in human MCF-7 breast cancer cells has been shown to arrest the growth of 80% of malignant cells. Absence of the threedimensional structure of hMTHFS has hampered the rational design and optimization of drug candidates. Wu and colleagues report the structures of the apo form of hMTHFS, hMTHFS bound with ADP, hMTHFS in complex with an N5-iminium phosphate reaction intermediate, and an enzyme-inhibitor complex of hMTHFS. Using the structures as a guide, the authors have mapped essential residues by site-directed mutagenesis. The snapshot of the N5-iminium phosphate–bound hMTHFS accounts for the first intermediate formed during the conversion of 5-formylTHF to 5,10-methenylTHF. The authors show how binding of the folate analogue 10-formyTHF displaces Y152, which moves away from Y83 in order to avoid steric clashes. As shown on the cover, this places Y152 in front of the channel connecting the ATP-binding site with the substrate binding pocket, obstructing any further nucleophilic attacks by the γ-phosphate of ATP. For details, see the article by Wu and colleagues on page 7294 of this issue.