A cryo light microscope with confocal/super-resolution capabilities to target macromolecular complexes for in-cell structural biology

结构生物学是研究大分子的三维（3D）结构的生物学领域，特别是蛋白质复合物，这是执行细胞功能并允许细胞生命的主要分子机器。了解蛋白质复合体的原子结构细节可以让科学家了解它的功能，因为3D结构显示了氨基酸在空间中的化学排列，这是解释蛋白质如何工作的关键。许多疾病都与蛋白质结构、蛋白质复合物组装、蛋白质-蛋白质相互作用、病原体蛋白质的缺陷有关，这使得了解蛋白质结构成为找到对抗疾病手段的基础。经典的结构生物学方法研究从细胞环境中提取的蛋白质复合物的结构，然而结构生物学的长期目标是解决蛋白质在细胞环境中的结构，这是它们最生理的环境。为了实现这一目标，必须在细胞环境中找到感兴趣的蛋白质，并且必须在同一区域切割薄片，以便在极低温度（-196℃）下用电子显微镜获得高分辨率图像。从这样的图像中可以获得一个3D体积，必须用计算方法提取蛋白质，以接近原子分辨率解决它们的3D结构。在细胞环境中找到感兴趣的蛋白质的方法之一是使用分子生物学方法将荧光标签附着在蛋白质上。由该提案资助的仪器将允许在-196℃下以高空间精度发现蛋白质荧光。在冷冻样品中如此精确的定位对于针对薄片（1米厚的1500亿分之一）的切割进行后续结构生物学表征至关重要。得到资助的仪器是一种尖端的光学显微镜，它可以高速成像感兴趣的细胞，而不会对生物样品造成任何损害。这台机器将使分子和细胞医学、感染和抗生素耐药性以及神经生物学等与MRC相关的领域的几个研究项目成为可能。该显微镜将用于了解哺乳动物和酵母细胞中涉及染色体分离的蛋白质复合物的结构（Barford实验室），了解细胞在每次分裂后如何允许染色体正确分布的基本细胞过程。因此，结构知识对于在这种蛋白质机制存在缺陷的情况下找到治疗靶点至关重要，因为这种缺陷是癌细胞的标志。该显微镜还将用于研究细菌细胞分裂的机制（Löwe lab），这是一个在所有微生物世界中保守的过程，当结构信息可用时，可以解决对抗感染性细菌的问题。巴拉特实验室将使用该仪器在分子水平上研究细菌生物膜的形成和组织，这是许多致病菌（如铜绿假单胞菌）感染的基本机制，铜绿假单胞菌是人类抗生素耐受性感染的主要原因，已被世界卫生组织列为研究新疗法的关键优先事项。Allegretti实验室将使用该仪器研究核重塑和修复机制的结构，这是允许细胞迁移的关键蛋白质复合物，但如果失调，它们有利于癌症转移进展，如果发生突变，它们会导致过早衰老。总之，获得这样一台仪器将极大地支持分子生物学实验室（LMB）的研究计划，该实验室拥有30多个用户，在机器获得后的第一年已经计划了9个项目。LMB的科学家们相信，这种显微镜将因此成为增强LMB结构生物学目标的基本工具

Structural biology is the field of biology which investigates the three-dimensional (3D) structures of macromolecules, in particular of protein complexes, the main molecular machines which perform cellular functions and allow cellular life. Knowing the structure of protein complexes with atomic details allows scientists to understand its function, since the 3D architecture shows the chemical arrangement of aminoacids in space, key to interpret how a protein works. Many diseases are linked to defects in protein architecture, protein complex assembly, protein-protein interactions, pathogen's proteins, making the understanding of protein structure fundamental to find means to fight diseases. Classic structural biology methods study the structure of protein complexes extracted from their cellular context, nevertheless the long-term goal of structural biology is to solve the structure of proteins in their cellular milieu, their most physiological environment. In order to achieve such goal, the protein of interest must be found within the cellular environment and thin sections must be cut in the same area to acquire high-resolution images with an electron microscope at very low temperatures (-196C). From such images a 3D volume can be obtained from where proteins must be extracted with computational methods to solve their 3D structure at near atomic resolution.One of the ways to find the protein of interest in the cellular context is to use a fluorescent tag attached to the protein using molecular biology methods. The instrument funded by this proposal will allow to spot the protein fluorescence with high spatial precision at -196C. Such precise localisation in the frozen sample is fundamental to target the cut of a thin section (150 billionth of a meter thick) for subsequent structural biology characterisation. The instrument to be funded is a cutting-edge light microscope which allow to image the cell of interest at high speed and without provoking any damage to the biological sample. Several research projects will be made possible from this machine, in the area of molecular and cellular medicine, infection and antibiotic resistance, and neurobiology, all relevant areas to the MRC. The microscope will be used to understand the structure of the protein complexes involved in chromosomes segregation in mammalian and yeast cells (Barford lab), fundamental cellular process to understand how a cell allows a correct distribution of chromosomes after each division. The structural knowledge will be therefore critical to find therapeutic targets in case of defects in this protein machinery, since such defects are a hallmark of cancer cells. The microscope will be also used to investigate the mechanism of bacterial cell division (Löwe lab), a conserved process in all microbial world that can be tackled to fight infectious bacteria when structural information will be available.Bharat lab will instead use the instrument to investigate the formation and organisation of bacterial biofilms at the molecular level, a fundamental mechanism of infection used by many pathogenic bacteria such as P. aeruginosa, which is a major cause of antibiotic-tolerant infections in humans and has been listed as a critical priority for the research of new treatments by the World Health Organisation.Allegretti lab will use the instrument to investigate the architecture of nuclear remodelling and repair's machineries, critical protein complexes that allow cell migration, but if dysregulated they favour cancer metastatic progression and, if mutated, they cause premature ageing. In summary the acquisition of such an instrument will tremendously support the research plans of the Laboratory of Molecular Biology (LMB) with more than 30 users and already 9 projects planned in the first year after machine's acquisition. LMB scientists are confident that this microscope will become therefore a fundamental tool to enhance the structural biology goals of the LMB