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射线晶体学来完成，其精度足以揭示单个原子的位置。然而，对于某些蛋白质，很难获得所需的晶体-因为难以产生足够量的蛋白质，或者因为分子本身难以结晶。重要的例子是膜蛋白的大类，其代表约30%的所有蛋白质和60%的药物靶点，但只有1%的已知结构。我们正在开发新的技术，以促进基于替代方法的结构测定-冷冻电子显微镜-它只需要非常少量的蛋白质，没有三维晶体。分子结构可以从电子显微镜图像中确定，这些图像是数千个嵌入在一层薄薄的冰层中的分子。这些图像被平均以提高其质量，并且通过比较不同取向的分子的图像来获得分子的三维结构-使用随机取向分子的样品或通过倾斜样品获得。结果主要取决于样品的质量-它有多平？冰的厚度有多均匀？能识别多少分子相邻分子的图像会互相干扰吗？我们正在开发一种新的方法来制备蛋白质样品的冷冻电子显微镜，有可能增加其作为一种工具，用于确定难以结晶的分子的结构的有用性。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10纳米左右的间距以规则模式结合靶分子。这就产生了一个密集的、平坦的非重叠分子阵列，这是结构测定的理想选择。我们已经对一种结构未知的膜蛋白进行了初步研究，并表明我们的技术可以获得优异的结果。我们将在研究其他结构未知的重要蛋白质的同时开发这项技术。我们的目标是开发一种普遍适用的技术，可以通过其他团体，以提高质量的结构信息，从低温电子显微镜和技术的吞吐量。

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.