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Cryo-electron microscopy using DNA-templated protein arrays

使用 DNA 模板蛋白质阵列的冷冻电子显微镜

基本信息

批准号：
BB/H000321/1
负责人：
Andrew Turberfield
金额：
$ 62.14万
依托单位：
University of Oxford
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2009
资助国家：
英国
起止时间：
2009 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FH000321%2F1
关键词：
Cryo electron microscopy using DNA

项目摘要

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.

由蛋白质和 RNA 制成的分子机器、信号分子和结构成分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，具有足够的精度来揭示单个原子的位置。然而，对于某些蛋白质来说，很难获得所需的晶体——要么是因为难以生产足够数量的蛋白质，要么是因为该分子本身就难以结晶。重要的例子是一大类膜蛋白，它们约占所有蛋白质的 30% 和 60% 的药物靶标，但仅占已知结构的 1%。我们正在开发新技术，以促进基于替代方法（冷冻电子显微镜）的结构测定，该方法只需要非常少量的蛋白质，并且不需要三维晶体。分子结构可以通过嵌入薄冰层中的数千个分子的电子显微镜图像来确定。对这些图像进行平均以提高其质量，并且通过比较不同方向的分子图像来获得分子的三维结构 - 使用随机方向的分子样本或通过倾斜样本获得。结果很大程度上取决于样本的质量——样本的平坦程度如何？冰层厚度如何均匀？可以识别多少个分子？相邻分子的图像会相互干扰吗？我们正在开发一种为冷冻电子显微镜制备蛋白质样品的新方法，这有可能提高其作为确定难以结晶分子结构的工具的实用性。我们的方法基于使用由合成 DNA 制成的自组装晶格，该晶格提供锚定点以规则图案结合目标分子，间距约为 10 纳米。这会产生密集、平坦的非重叠分子阵列，非常适合结构测定。我们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优异的结果。我们将在研究结构未知的其他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，可供其他小组采用，以提高冷冻电子显微镜获得的结构信息的质量和该技术的吞吐量。

项目成果

期刊论文数量（9）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

DNA origami signposts for identifying proteins on cell membranes by electron cryotomography.

DOI：
10.1016/j.cell.2021.01.033
发表时间：
2021-02-18
期刊：
Cell
影响因子：
64.5
作者：
Silvester E;Vollmer B;Pražák V;Vasishtan D;Machala EA;Whittle C;Black S;Bath J;Turberfield AJ;Grünewald K;Baker LA
通讯作者：
Baker LA

An engineered dimeric protein pore that spans adjacent lipid bilayers.