Cryo-electron microscopy using DNA-templated protein arrays

分子机器、信号分子和由蛋白质和RNA组成的结构组件是生物学最重要的基本组成部分。研究它们功能的一种方法是确定它们的结构--这通常可以通过X射线结晶学来完成，其精度足以揭示单个原子的位置。然而，对于一些蛋白质来说，很难获得所需的晶体--要么是因为很难产生足够数量的蛋白质，要么是因为分子本身就很难结晶。重要的例子是一大类膜蛋白，它们约占所有蛋白质的30%，占药物靶标的60%，但只占已知结构的1%。我们正在开发一种新的技术，以促进基于另一种方法-冷冻电子显微镜-的结构确定，这种方法只需要非常少量的蛋白质，不需要三维晶体。从电子显微镜图像中可以确定嵌入在一层薄冰中的数千个分子的分子结构。对这些图像进行平均以提高它们的质量，通过比较分子在不同方向上的图像-要么使用随机取向的分子样本，要么通过倾斜样本获得分子的三维结构。结果在很大程度上取决于样品的质量--它有多平？冰层的厚度有多大？能辨认出多少分子？相邻分子的图像会相互干扰吗？我们正在开发一种新的方法来制备用于冷冻电子显微镜的蛋白质样品，这有可能增加其作为确定难以结晶的分子结构的工具的有用性。我们的方法是基于使用由合成DNA制成的自组装晶格，这些晶格提供锚定，以大约10纳米的间距以规则的图案结合目标分子。这创造了一种密集的、扁平的非重叠分子阵列，这是结构确定的理想选择。我们已经对一种以前未知结构的膜蛋白进行了初步研究，并表明我们的技术可以取得很好的结果。我们将在研究其他结构未知的重要蛋白质的同时发展这项技术。我们的目标是开发一种普遍适用的技术，可以被其他小组采用，以提高低温电子显微镜提供的结构信息的质量和该技术的产量。

Molecular machines, signalling molecules and structural components made from protein and RNA are some of the most important fundamental building blocks of biology. One way to study their function is to determine their structure - this can often be done by X-ray crystallography with sufficient accuracy to reveal the positions of individual atoms. For some proteins, however, it is difficult to obtain the required crystals - either because it is difficult to produce a sufficient quantity of the protein, or because the molecule is inherently difficult to crystallize. Important examples are the large class of membrane proteins which represent about 30% of all proteins and 60% of drug targets but only 1% of known structures. We are developing new techniques to facilitate structure determination based on an alternative method - cryo-electron microscopy - which requires only very small quantities of protein and no three-dimensional crystals. Molecular structures can be determined from electron microscope images of thousands of molecules embedded in a thin layer of ice. These images are averaged to improve their quality, and the three-dimensional structure of the molecule is obtained by comparing images of the molecule in different orientations - either using a sample of randomly oriented molecules or obtained by tilting the sample. The results depend critically on the quality of the sample - how flat is it? how even is the ice thickness? how many molecules can be identified? do images of neighbouring molecules interfere with each other? We are developing a new way to prepare protein samples for cryo-electron microscopy which has the potential to increase its usefulness as a tool for determining the structures of hard-to-crystallize molecules. Our method is based on the use of self-assembled lattices made from synthetic DNA that provide anchorages to bind the target molecules in a regular pattern with a spacing of around 10 nanometres. This creates a dense, flat array of non-overlapping molecules which is ideal for structure determination.We have carried out pilot studies on a membrane protein with previously unknown structure, and have shown that our techniques can achieve excellent results. We will develop this technique while studying other important proteins whose structures are unknown. Our goal is to develop a generally applicable technique that can be adopted by other groups to improve the quality of structural information available from cryo-electron microscopy and the throughput of the technique.