Time-resolved methodologies to provide both spatial and temporal resolution in Electron Microscopy

结构生物学一直是解开蛋白质内突变变化秘密的关键，并为治疗干预提供了平台。传统上，X射线结晶学提供了所需的高分辨率信息，但它依赖于有序的晶体，这限制了它的范围，特别是对于膜蛋白和大的蛋白质复合体。然而，这些系统需要进行结构性调查。或者，电子显微镜(EM)非常适合于研究蛋白质复合体和膜蛋白，但传统上受到较低分辨率的阻碍。然而，在更稳定的显微镜、先进的处理算法、快速的自动数据收集和提供更高质量图像的新型直接电子探测器的推动下，EM经历了一场“分辨率革命”。因此，非对称蛋白质的原子分辨正在成为标准，以前很难处理，但重要的医学靶点现在可以使用EM衍生结构进行基于结构的药物设计。EM的一个强大但未被开发的优势是能够捕获存在于样品中的不同构象状态。然而，这目前依赖于对存在于样品中的“基态”中的不同构象状态的计算分类。要克服这一点，可以使用时间分辨方法。这种方法依赖于在冷冻之前将蛋白质和底物快速混合在液体冷冻剂中，以在ms时间范围内捕获反应中间产物。重要的挑战是，要用电子显微镜为蛋白质成像，必须将其冻结在玻璃冰中的特殊网格上，玻璃冰足够薄，电子可以通过，但不能比正在研究的样品薄。这通常是通过使用自动“吸墨”设备(如Vitbot)来实现的，该设备从网格中吸去多余的溶液，留下适量的溶液，以便在快速冻结时形成适当厚度的玻璃冰层。我们进行时间分辨研究的主要途径将是在微流控室中快速混合蛋白质和底物，然后直接将其喷洒到栅格上并进行冷冻。我们有一个基本的设置，能够分辨50毫秒的时间，但方法仍处于初级阶段，尚未充分发挥其潜力。这受到两个主要因素的限制，第一是在获得合适的冰层厚度方面与直接喷洒方法的不一致。通过筛选一系列网格类型，包括最近开发的微纤维网格，我们有信心找到一个可行的解决方案。第二个限制是在获得可靠、可重复和快速的快速混合设备方面。通过与汉堡大学的一项新合作，我们将把为尖端衍射技术(X射线、自由电子激光和SAXS)设计的技术转化为开发快速混合单元。通过与领先的网格准备系统(Vitbot)集成，我们可以开发一个可供任何感兴趣的EM小组访问的通用系统，从而将这项技术从利基市场推向主流。这笔资金将允许我们朝着这一目标迈出重要的第一步，通过生成一个能够以更可控和更可靠的方式产生时间分辨率&gt；5ms的原型系统。展望未来，结构生物学的下一个重要里程碑将是捕捉时间和空间分辨率，极大地增加我们对蛋白质和蛋白质复合体的理解，使我们摆脱目前该领域占主导地位的“静态”观点。

Structural biology has been key to unlocking the secrets of mutational change within proteins and provides the platform for therapeutic intervention. Traditionally, X-ray crystallography has provided the high resolution information required, but it is dependent on well-ordered crystals, limiting its scope, especially for membrane proteins and large protein complexes. Yet these systems demand structural investigation. Alternatively, electron microscopy (EM) is ideally suited to studying protein complexes and membrane proteins but has traditionally been hindered by a more modest resolution. However, EM has undergone a "resolution revolution" driven by more stable microscopes, advanced processing algorithms, rapid automatic data collection and new direct electron detectors which offer greater quality images. As a consequence atomic resolution of non-symmetric proteins is becoming the norm and previously intractable but important medical targets are now amenable to structure based drug design using EM derived structures. A powerful but under exploited advantage of EM is the ability to trap different conformational states that exist within the sample. However, this is currently reliant on computational sorting of different conformational states that exist within the sample in the "ground state". To overcome this, time-resolved methodologies can be used. This methodology relies on mixing a protein and substrate rapidly before freezing in a liquid cryogen to trap the reaction intermediate in the ms time frame. The significant challenge is that to image a protein with the electron microscope it must be frozen on a specialised grid in vitreous ice which is thin enough for the electrons to pass through but not thinner than the sample being studied. This is commonly achieved using an automated "blotting" device (such as the Vitrobot), which blots away excess solution from the grid leaving a suitable amount such that upon rapid freezing a vitreous ice layer is formed of an appropriate thickness. Our primary route for time-resolved studies will be to rapidly mix protein and substrate in a microfluidic chamber before directly spraying it onto a grid and plunge freezing. We have a basic setup that is capable of a time resolution >50ms but methodologies are still in their infancy and have yet to realise their full potential. This is limited by two major factors, the first is in obtaining a suitable ice thickness with inconsistencies of the direct spraying approach. By screening a range of grid types including the recently developed micro-fibre grids that have been designed to negate this problem we are confident we can find a working solution. The second limitation is in obtaining a reliable, reproducible and fast rapid mixing device. Through a new collaboration with Hamburg University we will translate the technology designed for cutting edge diffraction technologies (x-ray free electron laser and SAXS) to develop a rapid mixing unit. By integrating with the leading grid preparation system (Vitrobot) we can develop a universal system accessible by any interested EM group, moving this technology from niche to mainstream. This funding will allow us to take the first significant step towards this by generating a prototype system capable of producing time resolution >5ms in a more controlled and reliable manner. Looking forward the next significant milestone in structural biology will be the capture of temporal as well as spatial resolution, greatly increasing our understanding of proteins and protein complexes and moving us away from the "static" view that currently predominates in the field.