Data availability: All raw movie frames, micrographs, the particle stack and relevant metadata files have been deposited into EMPIAR, with accession code EMPIAR-10330. The electron density map has been deposited into EMDB, with accession code EMD-20806. The model has been deposited in the PDB, with accession code 6UKJ. All data are available in the manuscript or the supplementary materials.

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Recent advances in single-particle cryogenic-electron microscopy have facilitated an exponential growth in the number of membrane protein structures determined to close to atomic resolution. Nevertheless, despite improvements in microscope hardware, cryo-EM software and sample preparation techniques, challenges remain for structural analysis of small-sized membrane proteins (i.e. < 150 kilodalton). Here we discuss recent examples of structures of macromolecules from this category determined by cryo-EM. We analyze the underlying difficulties, the enabling technologies such as the use of antibody fragments to gain size and provide fiducials for particle alignment, and the unresolved issues like dislocation of complexes at the air-water interface. Finally, we briefly highlight the biological relevance of some of these success stories, and our predictions for the future.

Multidrug-resistant Plasmodium falciparum parasites have emerged in Cambodia and neighboring countries in Southeast Asia, compromising the efficacy of first-line antimalarial combinations. Dihydroartemisinin + piperaquine (PPQ) treatment failure rates have risen to as high as 50% in some areas in this region. For PPQ, resistance is driven primarily by a series of mutant alleles of the P. falciparum chloroquine resistance transporter (PfCRT). PPQ resistance was reported in China three decades earlier, but the molecular driver remained unknown. Herein, we identify a PPQ-resistant pfcrt allele (China C) from Yunnan Province, China, whose genotypic lineage is distinct from the PPQ-resistant pfcrt alleles currently observed in Cambodia. Combining gene editing and competitive growth assays, we report that PfCRT China C confers moderate PPQ resistance while re-sensitizing parasites to chloroquine (CQ) and incurring a fitness cost that manifests as a reduced rate of parasite growth. PPQ transport assays using purified PfCRT isoforms, combined with molecular dynamics simulations, highlight differences in drug transport kinetics and in this transporter’s central cavity conformation between China C and the current Southeast Asian PPQ-resistant isoforms. We also report a novel computational model that incorporates empirically determined fitness landscapes at varying drug concentrations, combined with antimalarial susceptibility profiles, mutation rates, and drug pharmacokinetics. Our simulations with PPQ-resistant or -sensitive parasite lines predict that a three-day regimen of PPQ combined with CQ can effectively clear infections and prevent the evolution of PfCRT variants. This work suggests that including CQ in combination therapies could be effective in suppressing the evolution of PfCRT-mediated multidrug resistance in regions where PPQ has lost efficacy.

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