Measuring, analysing and adjusting the aggregation of macromolecules in solution prior to structure determination

大约在过去的五年中，利用单粒子低温电子显微镜（cryogenic electron microscopy, cryoEM）以接近原子的分辨率来确定大分子（如蛋白质复合物和膜蛋白）的结构已经成为可能。这些发展增加了探索化学空间的需求，以便为冷冻电镜准备样品。生物物理方法，如动态光散射（DLS）和热移测定可以用于这种探索。该公司目前正寻求将其机器人技术（目前用作自动结晶液滴设置器）作为使用新软件对cryoEM样品进行高通量生物物理分析的分配器推向市场。但是，还需要提出关于探索化学空间的建议，而目前还缺乏这些建议。本A4I提案旨在通过列出一组或多组可以充当“屏幕”的解决方案来提供帮助。筛管将包括一组（例如，48种）在过去取得良好结果的化学溶液。在使用中，每种溶液将与新样品混合，遵循所有样品的恒定工作流程。目的是确定以有利的方式改变目标行为的试剂，通常是通过打破聚集。（这与蛋白质结晶中的筛选类似，但探索的化学空间和使用的测定方法将有所不同。）目的是为蛋白质结构测定找到合适的起始条件。这种新的工作流程将大大减少蛋白质结构测定所需的试验次数，特别是通过低温电镜，并增加结构生物学实验室的吞吐量。因此，它将为该公司的机器人平台在单颗粒冷冻电镜样品制备方面开辟一个新的市场。

In roughly the last five years it has become possible to determine the structures of macromolecules such as protein complexes and membrane proteins at near-atomic resolution using single particle cryogenic electron microscopy (cryoEM). These developments have increased the need to explore chemical space in order to get samples ready for cryoEM. Biophysical methods such as dynamic light scattering (DLS) and thermal shift assays can be used for this exploration. The company is now seeking to market its robotics -- currently used as automated crystallization drop-setters -- as dispensers for high throughput biophysical analysis of samples for cryoEM, using new software. However, recommendations for the exploration of chemical space will also be required, and these are, so far, lacking. This A4I proposal aims to help by listing one or more sets of solutions that can act as "screens". A screen will comprise a set of (e.g., 48) chemical solutions that have given favourable results in the past. In use, each solution would be mixed with a new sample, following a constant workflow for all samples. The objective is to identify reagents that alter the behaviour of the target in favourable ways, usually by breaking up aggregation. (This is similar to screening in protein crystallization, but the chemical space explored and the assay used will be different.) The objective is to find suitable starting conditions for protein structure determination. This new work-flow will dramatically reduce the number of trials needed for protein structure determination especially by cryoEM, and increase the throughput of structural biology labs. It will thus open a new market for the company's robotic platforms in the context of sample preparation for single particle cryoEM.