Towards higher resolution and cellular imaging using cryo-electron tomography

研究大分子络合物的结构对于了解它们的功能是必不可少的。此外，在细胞范围内研究大分子复合体的结构提供了关于其分子功能的最可靠的信息。冷冻电子断层扫描(CRYO-ET)是一种强大的成像技术，它提供了关于细胞结构的三维信息，没有化学固定和染色伪影。CRYO-ET的分辨率可达~10 nm，最高可达3 nm，并可在硅胶中实现亚层图像平均。电子剂量限制辐射损伤和厚样品是低温电子发射分辨的限制因素。这种分辨率提供了有关蛋白质整体形状的信息，但不能提供任何分子机制。我的研究重点是以微管二重体为测试样品，将低温等离子体的分辨率提高到亚纳米级。在这样的分辨率下，二级结构是清楚可识别的，因此，亚基的原子模型可以对接到实验断层扫描图中，以获得对蛋白质-蛋白质相互作用和分子机制的新见解。我的研究还将推动CRYO-ET在更厚的样品上工作，即通过使用相位板、直接电子探测器和改进的采集方案来提高信噪比来观察部分细胞。这将为我们提供一种独特的能力，打开一扇进入细胞的窗口，将细胞过程视为前所未有的分辨率。此外，为了解决细胞过程的动态本质，我的实验室将开发相关的光-电子显微镜技术，该技术结合了冷冻技术解析超微结构的能力和荧光显微镜的特定识别能力，以根据其周围环境精确定位感兴趣的蛋白质，从而深入了解基本的生物机制。*我们方法开发的测试样本将是真核纤毛，它存在于人类几乎所有的细胞中。我们计划使用CRYO-ET从纤毛中获得微管双重体的亚纳米结构，并使用Clem获得参与完整细胞内纤毛组装的鞭毛内转运复合体的原位结构。这里概述的技术发展将连接从分离的大分子络合物到体内细胞结构的多尺度分辨率。这项技术将为细胞的内部工作以及它们在疾病状态下如何改变提供重要的见解。我们的高分辨率结构将阐明微管二联体的生物物理性质背后的基本原理，并导致其功能的机制和潜在的生物材料研究的新思路。

Studying the structure of macromolecular complexes is essential for understanding how they function. Furthermore, studying the structure of the macromolecular complexes within their cellular context provides the most reliable information about their molecular function. Cryo-electron tomography (cryo-ET) is a powerful imaging technique that provides three-dimensional information about cellular architecture devoid of chemical fixation and staining artifacts. Cryo-ET can reach ~10 nm resolution and up to 3 nm with subtomogram averaging in silico. The limiting factor in cryo-ET resolution is the electron dose-limiting radiation damage and thick specimen. This resolution gives the information about the overall shape of the proteins but is not able to provide any molecular mechanism. My research focus is to push the resolution of cryo-ET to sub-nanometer resolution using the microtubule doublet as the test sample. At such resolutions, secondary structures are clearly identifiable and consequently, atomic models of subunits can be docked into the experimental tomographic maps to gain novel insights into protein-protein interactions and molecular mechanism. My research will also push cryo-ET to work on thicker sample i.e. looking at part of the cells by improving the signal-to noise ratio using the phase plate, direct electron detector and improved acquisition scheme. This will provide us with a unique ability to open a window into the cell to look at cellular process as unprecedented resolution. Furthermore, to address the dynamic nature of the cellular process, my laboratory will develop correlative light-electron microscopy technique, which combines the power of cryo-ET to resolve ultrastructure with the specific identification power of fluorescence light microscopy to precisely localize proteins of interest in the context of their surroundings to yield insights into fundamental biological mechanisms. ***The test sample for our method development will be the eukaryotic cilia, which are present in almost all cells in human. We plan to obtain the sub-nanometer structure of the microtubule doublet from the cilia using cryo-ET and the in situ structure of the intraflagellar transport complex, which is involved in the ciliary assembly within intact cells using CLEM. The technological developments outlined here will bridge multi-scale resolutions from isolated macromolecular complexes to in vivo cellular structures. The technology will provide important insights into the inner workings of cells and how they are altered in disease states. Our high-resolution structure will shed light onto the fundamental principals behind the biophysical properties of microtubule doublet, and lead to the mechanism of its function and potentially new ideas in biomaterial research.**