The discovery and design of biologically important RNA molecules is outpacing three-dimensional structural characterization. Here, we demonstrate that cryo-electron microscopy can routinely resolve maps of RNA-only systems and that these maps enable subnanometer-resolution coordinate estimation when complemented with multidimensional chemical mapping and Rosetta DRRAFTER computational modeling. This hybrid ‘Ribosolve’ pipeline detects and falsifies homologies and conformational rearrangements in 11 previously unknown 119- to 338-nucleotide protein-free RNA structures: full-length Tetrahymena ribozyme, hc16 ligase with and without substrate, full-length Vibrio cholerae and Fusobacterium nucleatum glycine riboswitch aptamers with and without glycine, Mycobacterium SAM-IV riboswitch with and without S-adenosylmethionine, and the computer-designed ATP-TTR-3 aptamer with and without AMP. Simulation benchmarks, blind challenges, compensatory mutagenesis, cross-RNA homologies and internal controls demonstrate that Ribosolve can accurately resolve the global architectures of RNA molecules but does not resolve atomic details. These tests offer guidelines for making inferences in future RNA structural studies with similarly accelerated throughput.

生物学上重要的 RNA 分子的发现和设计正在超越三维结构表征。在这里，我们证明冷冻电子显微镜可以常规地解析仅 RNA 系统的图谱，并且当与多维化学图谱和 Rosetta DRRAFTER 计算模型相补充时，这些图谱能够实现亚纳米分辨率的坐标估计。这种混合“Ribosolve”管道可检测和伪造 11 种以前未知的 119 至 338 核苷酸无蛋白质 RNA 结构中的同源性和构象重排：全长四膜虫核酶、有或没有底物的 hc16 连接酶、全长霍乱弧菌和具核梭杆菌含和不含甘氨酸的甘氨酸核糖开关适体、含和不含S-腺苷甲硫氨酸的分枝杆菌SAM-IV 核糖开关，以及含和不含 AMP 的计算机设计的 ATP-TTR-3 适体。模拟基准、盲目挑战、补偿性诱变、RNA 间同源性和内部对照表明 Ribosolve 可以准确解析 RNA 分子的全局结构，但不能解析原子细节。这些测试为未来 RNA 结构研究中的推断提供了指导，并具有类似的加速通量。