A next-generation energy filter for electron cryotomography at Imperial College

生命分子机器的功能与它们在细胞内的环境密切相关。许多多蛋白质复合体是动态的、脆弱的或膜相关的，这使得它们具有挑战性或不可能使用传统的结构生物学方法进行有效的研究，这些方法包括通过X射线结晶学、核磁共振或单颗粒分析低温电子显微镜对蛋白质进行纯化和表征。因此，在现场研究分子机器对于我们充分了解其机理是至关重要的，并将为21世纪生物科学的发展奠定基础。这些基础性工作还将应用于现场可视化疾病和病原体的机制、药物干预作用的细胞分支，以及合成设计(或现有分子机器的选择)。将分子机器的结构分子生物学就地可视化的唯一方法是使用电子冷冻断层扫描，它利用电子冷冻显微镜的现有能力(例如，与2017年诺贝尔化学奖)从一系列角度对独特的生物样本(如细胞)成像，以重建其三维结构并解析其分子成分：有效地进行一次“纳米级CT扫描”。这些3-D图像，或断层图像，解开了细胞的复杂性，否则在2-D图像中是压倒性的；类似的结构(例如，蛋白质复合体)随后可以被比对、分类和平均，以得出它们在情况下的三维结构快照。我们的建议是购买下一代“能量过滤器”，以实现伦敦帝国理工学院的常规电子冷冻断层扫描。此次收购将推动帝国能源通过一次升级采购从“落后于时代”转变为“具有国际竞争力”，并促进我们各种COI工作中已经萌芽的不同项目的发展。

The function of the molecular machines of life is intimately associated with their context inside cells. Many multiprotein complexes are dynamic, fragile, or membrane-associated, making them challenging or impossible to effectively study using the traditional structural biology approaches that involve protein purification and characterization by X-ray crystallography, nuclear magnetic resonance, or single particle analysis cryoEM. Studying molecular machines in situ is therefore crucial for us to fully understand their mechanisms and will form a foundation for the development of the biological sciences into the 21st century. Such foundational work will also have application in visualizing the mechanisms of diseases and pathogens in situ, the cellular ramifications of the action of pharmaceutical interventions, and the synthetic design (or co-option of existing) molecular machines. The only way to visualize the structural molecular biology of molecular machines in situ is by using electron cryotomography, which co-opts the existing power of electron cryomicroscopes (e.g., with the 2017 Nobel Prize in Chemistry) to image unique biological samples such as cells from a range of angles to reconstruct their 3-D structures and to resolve their molecular components: effectively a "nanoscale CT scan". These 3-D images, or tomograms, disentangle the complexity of the cell that would otherwise be overwhelming in a 2-D image; similar structures (e.g., protein complexes) can subsequently be aligned, classified, and averaged, to arrive at 3-D structural snapshots of their action in situ.Our proposal is to purchase a next generation "energy filter" to enable routine electron cryotomography at Imperial College London. This purchase will propel Imperial from being "behind the times" to "internationally competitive" with a single upgrade purchase and catalyse development of diverse projects already nascent in our various CoI's work.