In situ cryoET reveals inhibitor-induced perturbations in Pf80S GTPase interactions

Abstract Malaria parasites rely heavily on rapid, high fidelity protein synthesis to infect and replicate in human erythrocytes, making translation an attractive target for new antimalarials. Here, we have determined in situ structures of Pf 80S ribosomes in thirteen conformational and compositional states from cryoFIB-milled Plasmodium falciparum -infected human erythrocytes across the stages of asexual intraerythrocytic parasite replication. We observe eight active translation intermediates, enabling us to define the native malarial translation elongation cycle, which surprisingly features a bifurcation at the decoding stage of the cycle that has not previously been described. Examination of perturbations in the distribution of ribosomes among these states in the presence of a malaria-specific translation inhibitor suggests that the inhibitor impedes Pf eEF2 and Pf eEF1α interactions with the ribosome. We integrated our in situ cryoET data with proteomic and ultrastructural data to arrive at a deeper understanding of malarial translation, which will inform development of new therapies.