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

近五年来，利用单粒子低温电子显微镜(CryoEM)以近原子分辨率确定蛋白质复合体和膜蛋白质等大分子的结构已成为可能。这些发展增加了探索化学空间的需要，以便为低温EM准备样品。生物物理方法，如动态光散射(DLS)和热位移分析，可以用于这一探索。该公司现在正寻求将其机器人--目前被用作自动结晶滴定器--作为分配器，使用新软件对CryoEM样品进行高通量生物物理分析。然而，还需要就探索化学空间提出建议，而到目前为止，这些建议还没有。这份A4I提案旨在通过列出一套或多套可以充当“屏幕”的解决方案来提供帮助。筛选将包括一组(例如，48个)过去已产生有利结果的化学溶液。在使用中，每个解决方案将与一个新的样品混合，对所有样品遵循一个恒定的工作流程。目标是确定以有利的方式改变目标行为的试剂，通常是通过破坏聚集。(这类似于蛋白质结晶中的筛选，但探索的化学空间和使用的分析方法将有所不同。)目的是为蛋白质结构测定寻找合适的起始条件。这一新的工作流程将极大地减少蛋白质结构确定所需的试验次数，特别是通过低温EM，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粒子CryoEM样品制备方面打开一个新的市场。

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.