Enhancing the Site-directed RNA Editing Toolkit with Cryo-EM Structures of Native RNA Editing Complexes

成功地制备了蛋白质和两个RNA靶标，并通过EMSA的生化表征验证了复合体的形成。对ADAR2：Gli1-61BP的EMSA表征表明，当蛋白质与RNA的摩尔比为2：1时，与一个、两个或三个ADAR2单体结合的RNA之间存在平衡的络合物，其中大部分处于两个或三个ADAR2状态。选择2：1的比例进行低温电子显微镜数据采集。在泰坦Krios冷冻电子显微镜上收集了超过15,000张ADAR2：Gli1-61BP复合体的显微照片。在分离出良好的颗粒图像并进行3D重建后，发现冰中颗粒的角分布不足以进行准确的3D重建。为了增加角度分布，在开始数据收集之前，样品倾斜到30度角。在收集了大约3500张显微照片后，这项技术导致了ADAR2：Gli1-61bp在5角处的三维重建，其中可以清楚地看到蛋白质的二级结构。二级结构的区分使结构模型的准确分配成为可能，包括RNA、脱氨酶结构域和dsRBDS。EMSA对ADAR2：HT2cR-89聚体复合体的EMSA表征表明，当蛋白质与RNA的摩尔比为4：1时，复合体仅以与两个ADAR2分子结合的RNA形式存在。4：1的比例用于制备低温电子显微镜网格，但冷冻和数据收集条件仍在优化中。

Protein and both RNA targets have been successfully produced, and complex formation has been verified with biochemical characterization using EMSA. Data collection is ongoing.EMSA characterization of ADAR2:GLI1-61bp showed that at a 2:1 molar ratio of protein to RNA, complex exists in equilibrium between RNA bound by one, two, or three ADAR2 monomers, with a majority being in the two- or three-ADAR2 state. The 2:1 ratio was chosen for cryoEM data collection. Over 15,000 micrographs of ADAR2:GLI1-61bp complex were collected on the Titan Krios cryo-electron microscope. After isolation of good particle images and 3D reconstruction, the angular distribution of particles in the ice was found to be insufficient for accurate 3D reconstruction. To increase the angular distribution, the sample was tilted to a 30deg angle before beginning data collection. After collecting about 3500 micrographs, this technique resulted in a 3D reconstruction of ADAR2:GLI1-61bp at 5-angstroms in which protein secondary structures can clearly be seen. The distinguishment of secondary structures allowed for accurate assignment of structural models to the reconstruction, including the RNA, deaminase domain, and dsRBDs.EMSA characterization of the ADAR2:HT2cR-89mer complex showed that at a 4:1 molar ratio of protein to RNA, complex exists exclusively as RNA bound by two ADAR2 molecules. The 4:1 ratio was used for preparation of cryoEM grids, but freezing and data collection conditions are still being optimized.