High-throughput low-volume crystallisation facility

这是一项设备提案，以实现开创性的研究。我们对蛋白质和核酸(如DNA)如何工作的了解几乎都来自结构生物学。这需要生长这些大分子的晶体，就像Max Perutz的开创性工作一样，他因解决血液中携带氧气的蛋白质--血红蛋白的结构而在1962年获得诺贝尔奖。最难结晶的东西是位于活细胞周围细胞膜中的蛋白质，因为它们不溶于水--但这些蛋白质是所有药物的靶标。它们还能将阳光转化为能量，传导神经冲动，并将各种营养物质输送到细胞中。在过去的十年里，蛋白质结晶方法发生了一场革命，特别是膜蛋白质，我们的建议是为利兹大学以及当地其他大学(谢菲尔德、哈德斯菲尔德、纽卡斯尔和曼彻斯特)配备这种尖端设备。该设备有三个组成部分：(1)表征设备(SEC-MALLS，LCP-FRAP)，它将帮助我们确定蛋白质在所使用的条件下是否有可能结晶；(2)结晶机器人，这样我们在每次结晶试验中可以使用比以前少20-50倍的蛋白质(20-50 NL，而不是1微升)；以及(3)4摄氏度和室温下的机器人成像仪。由于我们将进行数以万计的实验，机器人成像器使实验可视化比在显微镜下逐个观察每个实验要容易得多。此外，晶化机器人还能使“脂立方相”(LCP)滴化，相当于一次挤出50毫升的牙膏。LCP特别革命性地改变了膜蛋白的结晶，但就像牙膏一样，它是不透明的。因此，我们还购买了一台“SONICC”成像仪，它将使我们能够在不透明的液晶中看到非常小的蛋白质晶体。后基因组时代为生命背后的化学和调控机制提供了意想不到的见解，结构生物学是其中非常重要的一部分。尽管在水溶性蛋白质方面取得了成功，但仍然存在重大挑战，特别是在膜蛋白和大型哺乳动物/真核蛋白质复合体方面，这一设备将解决这些问题。阿斯特伯里结构分子生物学中心的结构工作围绕着四个主要重叠的主题领域：(1)膜蛋白；(2)大复合体；(3)病原体-宿主相互作用；以及(4)小分子(即药物)的设计。我们期待突破的项目有：涉及感知疼痛、温度或味道的离子通道如何工作？像普通感冒或天花这样含有RNA的病毒是如何将RNA包装在自己体内的？这是病毒具有感染性所必需的。像液泡ATPase这样的大分子机器是如何工作的，它们是如何被调节的？这些在锥虫寄生虫中很重要，这种寄生虫会导致动物和人类的重大疾病。我们能更好地了解一些植物如何抵抗铝等金属的毒性吗？因此，我们能否提高主要农作物的这种能力？这将有助于作物在酸性土壤中以较少的化肥使用量生长得更好。最后，积聚在表面上的细菌会形成生物膜--牙齿周围、假体植入物周围、船舶表面，这会产生不利的后果。要了解这种情况是如何发生的并加以预防，需要了解相关蛋白质的结构。

This is an equipment proposal, to enable ground-breaking research. Almost all of our understanding of how proteins and nucleic acids such as DNA work has come from structural biology. This requires growing crystals of these macromolecules, as in the pioneering work of Max Perutz, who won a Nobel prize in 1962 for solving the structure of the oxygen-carrying protein of the blood, haemoglobin. The hardest things to crystallise are the proteins that sit in the membranes that surround living cells, because they are not soluble in water - but these proteins are the targets for 50% of all drugs. They are also the ones that turn sunlight into energy, conduct nerve impulses and transport nutrients of all kinds into cells.Over the last ten years, there has been a revolution in methods for crystallising proteins, especially membrane proteins, and our proposal is to equip the University of Leeds, and thus other local universities (Sheffield, Huddersfield, Newcastle and Manchester) with this cutting-edge equipment. The equipment has three components: (1) characterisation equipment (SEC-MALLS, LCP-FRAP), which will help us determine if the protein is likely to be crystallisable in the conditions being used; (2) a crystallisation robot so that we can use 20-50 times less protein than before ("drops" of 20-50 nl, rather than 1 ul) in each crystallisation trial; and (3) robotic imagers both at 4 C and room temperature. As we will be doing tens of thousands of trials, robotic imagers make visualising the experiments much easier than having to look at each experiment one by one under a microscope. In addition, the crystallisation robot can make "lipidic cubic phase" (LCP) drops, which corresponds to squeezing out 50 nl of toothpaste at a time. LCP has in particular revolutionised the crystallisation of membrane proteins but, like toothpaste, it is opaque. Consequently, we are also buying a "SONICC" imager, which will enable us to see very small protein crystals in the opaque LCP.The post-genomic era has provided unimagined insights into the chemistry and regulatory mechanisms underlying life, and structural biology has been an very important part of this. Despite successes with water-soluble proteins, major challenges remain, particularly for membrane proteins and large mammalian/eukaryotic protein complexes, which this equipment will address. The structural work at the Astbury Centre for Structural Molecular Biology is centred around four major overlapping theme areas: (1) Membrane proteins; (2) large complexes; (3) pathogen-host interactions; and (4) design of small molecules (i.e. drugs).Examples of projects where we expect breakthroughs are: how do the ion-channels involved in sensing pain, temperature or taste work? How do viruses that contain RNA, like the common cold or smallpox, package the RNA inside themselves? This is required for the virus to be infective. How do large molecular machines, like the vacuolar ATPase, work and how are they regulated? These are important in trypanosomal parasites that cause major diseases in both animals and humans. Can we understand better how some plants resist the toxicity of metals such as aluminium, and can we therefore enhance this ability in major crops? This will help make crops grow better, with less use of fertilisers, in acidic soils. Finally, bacteria that accumulate on surfaces form biofilms - around teeth, around prosthetic implants, on the surfaces of ships, with adverse consequences. Understanding how this happens and preventing it requires understanding the structures of the proteins involved.