High-throughput low-volume crystallisation facility

这是一项设备提案，以实现突破性的研究。我们对蛋白质和核酸（如DNA）如何工作的理解几乎都来自结构生物学。这需要培养这些大分子的晶体，就像马克斯·佩鲁茨（Max Perutz）的开创性工作一样，他在1962年因解决血液中携带氧气的蛋白质——血红蛋白的结构而获得诺贝尔奖。最难结晶的是活细胞周围膜上的蛋白质，因为它们不溶于水——但这些蛋白质是50%的药物的目标。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，在结晶蛋白质，特别是膜蛋白的方法上发生了一场革命，我们的建议是为利兹大学以及其他当地大学（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），这将帮助我们确定在使用的条件下蛋白质是否可能结晶；(2)一个结晶机器人，这样我们就可以在每次结晶试验中使用比以前少20-50倍的蛋白质（“滴”20-50 nl，而不是1 ul）；(3) 4℃和室温下的机器人成像仪。由于我们将进行成千上万的试验，机器人成像仪使实验可视化比在显微镜下一个接一个地观察每个实验要容易得多。此外，结晶机器人可以制造“脂质立方相”（LCP）液滴，相当于一次挤出50升牙膏。LCP尤其彻底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“SONICC”成像仪，这将使我们能够在不透明的LCP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，而结构生物学是其中非常重要的一部分。尽管在水溶性蛋白方面取得了成功，但主要的挑战仍然存在，特别是对于膜蛋白和大型哺乳动物/真核蛋白复合物，该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四个主要重叠的主题领域展开：(1)膜蛋白；(2)大型综合体；(3)病原-宿主相互作用；(4)小分子（即药物）的设计。我们期待突破的项目有：参与感知疼痛、温度或味觉的离子通道是如何工作的？含有RNA的病毒，比如普通感冒病毒或天花病毒，是如何将RNA包装在自身内部的？这是病毒具有传染性的必要条件。像液泡atp酶这样的大分子机器是如何工作的？它们是如何被调节的？这些对在动物和人类中引起重大疾病的锥虫体寄生虫很重要。我们能否更好地理解一些植物是如何抵抗铝等金属的毒性的？我们能否因此提高主要作物的这种能力？这将有助于作物在酸性土壤中以更少的肥料生长得更好。最后，细菌在牙齿周围、假体周围、船舶表面积聚形成生物膜，造成不良后果。了解这种情况是如何发生并预防的，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.