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

蛋白质和RNA靶点已成功生产，并通过EMSA进行生化表征验证了复合物的形成。数据收集正在进行中。EMSA对ADAR2:GLI1-61bp的表征表明，当蛋白质与RNA的摩尔比为2:1时，复合物在RNA与一个、两个或三个ADAR2单体结合的平衡状态下存在，其中大多数处于两个或三个ADAR2状态。冷冻电镜数据采集采用2:1的比例。在Titan Krios冷冻电子显微镜上收集了超过15,000张ADAR2:GLI1-61bp复合物的显微照片。在分离出良好的颗粒图像并进行三维重建后，发现冰中颗粒的角度分布不足以进行精确的三维重建。为了增加角度分布，在开始数据收集之前，将样本倾斜至30度角。在收集了大约3500张显微照片后，该技术在5埃下对ADAR2:GLI1-61bp进行了三维重建，可以清楚地看到蛋白质的二级结构。二级结构的区分允许准确分配结构模型的重建，包括RNA，脱氨酶结构域和dsrbd。EMSA对ADAR2:HT2cR-89mer复合物的表征表明，在蛋白质与RNA的摩尔比为4:1时，复合物仅以RNA形式存在，由两个ADAR2分子结合。冷冻电镜栅格的制备采用4:1的比例，但冷冻和数据采集条件仍在优化中。

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.