Faculty of Applied Sciences, Delft University of Technology, Delft, The Netherlands.
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
Sci Rep. 2023 Aug 16;13(1):13327. doi: 10.1038/s41598-023-39829-5.
Single molecule localization microscopy offers resolution nearly down to the molecular level with specific molecular labelling, and is thereby a promising tool for structural biology. In practice, however, the actual value to this field is limited primarily by incomplete fluorescent labelling of the structure. This missing information can be completed by merging information from many structurally identical particles in a particle fusion approach similar to cryo-EM single-particle analysis. In this paper, we present a data analysis of particle fusion results of fluorescently labelled Nup96 nucleoporins in the Nuclear Pore Complex to show that Nup96 occurs in a spatial arrangement of two rings of 8 units with two Nup96 copies per unit giving a total of 32 Nup96 copies per pore. We use Artificial Intelligence assisted modeling in Alphafold to extend the existing cryo-EM model of Nup96 to accurately pinpoint the positions of the fluorescent labels and show the accuracy of the match between fluorescent and cryo-EM data to be better than 3 nm in-plane and 5 nm out-of-plane.
单分子定位显微镜通过特定的分子标记提供接近分子水平的分辨率，因此是结构生物学的一种有前途的工具。然而，在实践中，由于结构的荧光标记不完全，其对该领域的实际价值主要受到限制。通过类似于 cryo-EM 单颗粒分析的颗粒融合方法，将许多结构相同的颗粒的信息融合，可以完成缺失的信息。在本文中，我们对荧光标记的核孔复合物中核孔蛋白 Nup96 的颗粒融合结果进行了数据分析，结果表明 Nup96 以 8 个单元的两个环的空间排列存在，每个单元有两个 Nup96 拷贝，每个核孔共有 32 个 Nup96 拷贝。我们使用人工智能辅助建模的 AlphaFold 来扩展现有的 Nup96 cryo-EM 模型，以准确确定荧光标记的位置，并表明荧光和 cryo-EM 数据之间的匹配精度在平面内优于 3nm，在平面外优于 5nm。
