Crystallization robotics to support X-ray crystallography at MUSC

描述(由申请人提供)： 随着改进的分相方法的出现，使用X射线结晶学确定大分子结构的限速步骤通常是结晶。结晶的目标是找到最佳的参数组合，以产生足够质量的晶体，以便它们可以用于产生高分辨率的衍射数据。这种搜索包括测试几乎无限数量的参数，包括蛋白质浓度、缓冲液的类型及其pH值、沉淀剂的性质和浓度、温度以及添加剂或配体的使用。因此，“结晶空间”是巨大的，如果用人工输送晶体屏幕的低通量方法来搜索，这个过程会受到可以提纯的生物材料数量的严重限制。配药机器人的应用可以极大地改变这种情况，因为它们可以吸管纳升体积的蛋白质，因此可以用更少的生物材料对条件进行更大程度的采样。托盘的设置速度也比手动方法快得多。尽管MUSC在X射线结晶学方面有一个正在发展和蓬勃发展的项目，包括蛋白质生产的共享资源，但我们制造晶体结构的能力因缺乏结晶机器人而严重受阻。这项申请旨在解决从Rigaku Inc.申请菲尼克斯RE纳升分配机器人的不足。这不仅将提高MUSC现有结晶学项目的生产率，还将刺激涉及更多研究人员的新活动，这些研究人员渴望使用X射线结晶学，但由于缺乏晶体而无法这样做。广泛的好处将是：(A)解决包括膜蛋白在内的更具挑战性和潜在回报的项目的能力；(B)由于更加重视对重要生物医学问题的合作机制研究，并可在影响较大的期刊上发表，因此改善了MUSC的研究环境；(C)提高了NIH资助的竞争力；(D)改善了研究生和博士后的培训环境；以及(E)通过招聘新教师，进一步发展了X射线结晶学和一般结构生物学。 与公共健康相关：该提案要求提供资金购买结晶机器人，以增强支持南卡罗来纳医科大学生物医学研究的X射线结晶学设施。X射线结晶学是一种确定大分子结构细节的方法，这些大分子的生物学功能可能对人类健康和疾病具有重要意义。对这些大分子的结构和功能的准确了解有助于更好地了解正常和异常的生物过程，并有助于开发改进的治疗方法来治疗或治愈癌症、传染病、炎症性疾病和阿尔茨海默病等重大疾病。

DESCRIPTION (provided by applicant): With the advent of improved methods for phasing, the rate-limiting step to determine the structure of a macromolecular using X-ray crystallography is most often crystallization. The goal of crystallization is to find the best combination of parameters to produce crystals of sufficient quality so they can be used to generate high resolution diffraction data. Such a search involves testing an almost limitless number of parameters, including protein concentration, type of buffer and its pH, nature and concentration of precipitant, temperature, and use of additives or ligands. Hence, "crystallization space" is immense and if searched by low-throughput methods where crystal screens are pipetted manually, the process is severely limited by the amount of biological material that can be purified. The application of dispensing robots can dramatically change this situation because these can pipette protein in nanoliter volumes and therefore allow a much greater sampling of conditions with less biological material. Trays can also be set up considerably faster than manual methods. Although MUSC has a developing and thriving program in X-ray crystallography, including shared resources for protein production, our ability to produce crystal structures is severely hampered by the lack of crystallization robotics. This application aims to address that deficiency requesting the Phoenix RE nanoliter dispensing robot from Rigaku Inc. This will not only enhance the productivity of existing crystallography projects at MUSC, but will also stimulate new activity involving a wider group of investigators who aspire to use X-ray crystallography and have been unable to do so for lack of crystals. The broad benefits will be (a) the ability to tackle more challenging and potentially rewarding projects, including membrane proteins, (b) enhancement of the research environment at MUSC due to a greater emphasis on collaborative mechanistic studies of important biomedical problems that can be published in the higher impact journals, (c) increased competitiveness for NIH funding, (d) improved training environment for graduate students and post-docs and, (e) further development of X-ray crystallography and of structural biology in general through recruitment of new faculty. PUBLIC HEALTH RELEVANCE: This proposal requests funds to purchase crystallization robotics to enhance the X-ray crystallography facility that supports biomedical research at the Medical University of South Carolina. X-ray crystallography is a method of determining the structural details of macromolecules whose biological functions may be significant in human health and disease. Precise knowledge of the structure and function of such macromolecules contributes to better understanding of normal and abnormal biological processes and development of improved therapies to treat or cure major illnesses such as cancer, infectious diseases, inflammatory diseases and Alzheimer's disease.