The diamide insecticide class is one of the top-selling insecticides globally. They are used to control a wide range of pests by targeting their ryanodine receptors (RyRs). Here, we report the highest-resolution cryo-electron microscopy (cryo-EM) structure of RyR1 in the open state, in complex with the anthranilic diamide chlorantraniliprole (CHL). The 3.2-Å local resolution map facilitates unambiguous assignment of the CHL binding site. The molecule induces a conformational change by affecting the S4–S5 linker, triggering channel opening. The binding site is further corroborated by mutagenesis data, which reveal how diamide insecticides are selective to the Lepidoptera group of insects over honeybee or mammalian RyRs. Our data reveal that several pests have developed resistance via two mechanisms, steric hindrance and loss of contact. Our results provide a foundation for the development of highly selective pesticides aimed at overcoming resistance and therapeutic molecules to treat human myopathies.

二酰胺类杀虫剂是全球最畅销的杀虫剂之一。它们可通过靶向其ryanodine受体（RyRs）来控制各种害虫。在这里，我们报告的高分辨率RyR1的冷冻电子显微镜（cryo-EM）结构处于开放状态，与邻氨基苯甲酰胺二甲基氯吡咯烷酮（CHL）配合使用。3.2-Å的局部分辨率图有助于CHL结合位点的明确分配。该分子通过影响S4–S5接头来诱导构象变化，从而触发通道开放。诱变数据进一步证实了结合位点，诱变数据揭示了二酰胺杀虫剂对蜜蜂或哺乳动物RyRs的鳞翅目昆虫的选择性。我们的数据表明，几种害虫通过两种机制（空间位阻和失去接触）产生了抗药性。