300 keV FEG Electron Cryo Microscope for Single Particle Analyses

单粒子电子冷冻显微镜（cryoEM）可以对细胞的大分子组成进行结构分析。探索结构-功能关系对于理解生物学中的基本分子机制至关重要。我们在这里的目标是详细分析大分子及其在感染过程中的动态变化-这是汉堡的主要研究重点。近年来的技术进步和突破已经使该技术达到了一种状态，允许在2.5和6Å之间的分辨率范围内实现稳定蛋白质复合物的常规三维结构。这一“革命”基于以下因素：(1)开发出具有平行照明和机器人样品处理功能的300keV高分辨率电子冷冻显微镜；(2)引入直接电子探测器；(3)自动化数据采集软件；(4)更强大和通用的分析软件包；(5)更好的计算硬件。总之，这些发展已经将单粒子低温电子显微镜移动到一个高效和结晶独立的生物大分子高分辨率结构分析的替代方案。特别是对于具有各种功能构象的更大更灵活的大分子复合物，单粒子冷冻电镜结合鲁棒分类算法在结构生物学中提供了一种非常有吸引力的方法。对冷冻电镜的需求已经大大增加，现在包括了“经典”结构生物学家以外的社区，如生物化学家和细胞生物学家（参见该仪器的广泛用户基础）。

Single particle electron cryo microscopy (cryoEM) enables structural analyses of the macromolecular building blocks of cells. Probing structure-function relationships is essential for understanding basic molecular mechanisms in biology. We here aim to analyse in detail macromolecules and their dynamic changes in the course of infections – a major research focus in Hamburg. The technical advancements and break troughs of the recent years have moved the technology to a state allowing to achieve routinely three-dimensional structures in a resolution range between 2.5 und 6Å for stable protein complexes. This ‚revolution’ is based on the following factors: (1) development of robust 300keV high-resolution electron cryo microscopes with parallel illumination and robotic sample handling, (2) introduction of direct electron detectors, (3) automated data acquisition software, (4) more robust and versatile analysis software packages and (5) better computing hardware. Together these developments have moved single particle cryoEM to an efficient and crystallisation independent alternative for high-resolution structural analyses of biological macromolecules. In particular for larger more flexible macromolecular complexes with various functional conformations single particle cryoEM in combination with robust classification algorithms provides a highly attractive approach in structural biology. Demand for respective cryoEM access has risen immensely and includes now communities beyond ‘classical’ structural biologists, like biochemist and cell biologists (cf. the broad user base of the instrument).