Ultraviolet (UV) light microscope for protein and nucleic acid crystal detection

大分子X射线结晶学提供了对生命分子基础的无与伦比的洞察，因为它是一种可以用来在原子分辨率或接近原子分辨率的情况下可视化蛋白质和核酸等生物分子的三维结构的方法。在过去的10年里，马尼托巴大学在该领域发展了强大的影响力，其中包括马克、斯特费尔德和麦肯纳博士的研究密集型实验室。我们的实验室共有29名HQP和受训人员，他们研究的课题从宿主-病原体相互作用的结构生物学、抗生素耐药性到基质生物学和叶绿素生物合成。 任何生物X射线结晶学研究项目成功的关键是在结晶实验中识别目标蛋白质、核酸或这些分子的更大的四元络合物的晶体的能力。通常必须使用立体显微镜对数百种化学条件进行筛选，以确定目标大分子或四元络合物结晶的少数条件。生长的晶体通常非常小(微晶体)，对比度低；因此，当使用传统的明场照明技术时，它们很容易被忽略。然而，用紫外线(UV)照明很容易克服这个问题，因为它显著地增强了蛋白质和/或核酸晶体的存在，因为它能够直接看到蛋白质和核酸晶体的固有色氨酸荧光和紫外吸收。当晶体生长在通常在结晶实验中形成的致密沉淀中时，这一点尤其有用。此外，紫外光显微镜可以用来区分蛋白质和/或核酸的晶体与盐的晶体，后者在结晶实验中经常出现，可能具有极大的误导性。鉴于紫外光显微镜的显著优势，它已成为现代生物X射线结晶学研究的重要组成部分。不幸的是，马尼托巴省没有设计用来在结晶实验期间检测蛋白质或核酸晶体的紫外光显微镜。这使马尼托巴省的结构生物学家和他们的HQP受训人员处于明显的劣势，限制了他们推进大分子结构研究的能力，这些大分子产生的微晶体即使不是不可能，也很难用传统的光学显微镜检测到。为了克服这一重大限制，我们请求为价格合理的紫外光显微镜提供资金，这将从根本上提高我们晶体筛选活动的成功和我们HQP培训的质量。该显微镜将集成到我们在马尼托巴大学的X射线结晶学设施中，并填补维持我们的研究和HQP培训能力所需的关键技术空白。

Macromolecular X-ray crystallography provides unparalleled insight into the molecular basis of life, since it is a method that can be used to visualize the three-dimensional structure of biomolecules such as proteins and nucleic acids at or near atomic resolution. The University of Manitoba has developed a strong presence in the field over the past 10 years, which includes the research-intensive laboratories of Drs. Mark, Stetefeld and McKenna. Together, our laboratories are staffed by 29 HQP and trainees who investigate topics ranging from the structural biology of host-pathogen interactions and antibiotic resistance to matrix biology and chlorophyll biosynthesis. Key to the success of any biological X-ray crystallography research program is the ability to identify crystals of target proteins, nucleic acids, or larger quaternary complexes of these molecules in crystallization experiments. Hundreds of chemical conditions must typically be screened using a stereo microscope to identify the few conditions in which a target macromolecule or quaternary complex will crystalize. Crystals that do grow are often very small (microcrystals) and have low contrast; thus, they are easily missed when using conventional brightfield illumination techniques. Illumination with ultraviolet (UV) light, however, readily overcomes this problem since it markedly enhances the presence of protein and/or nucleic acid crystals by enabling direct visualization of the intrinsic tryptophan fluorescence and UV absorption of protein and nucleic acid crystals, respectively. This is especially useful when crystals are growing within dense precipitates that often form in crystallization experiments. Moreover, UV light microscopy can be used to distinguish crystals of protein and/or nucleic acid from those of salt, the latter of which often arise in crystallization experiments and can be highly misleading. Given the remarkable advantages of UV light microscopy, it has become a vital part of modern biological X-ray crystallography research. Unfortunately, there is no UV light microscope in the province of Manitoba that is designed to detect protein or nucleic acid crystals during crystallization experiments. This puts Manitoba structural biologists and their HQP trainees at a distinct disadvantage by restricting their ability to advance structural studies of macromolecules that yield microcrystals which are very difficult, if not impossible, to detect by conventional light microscopy. To overcome this significant limitation, we request funding for a reasonably priced UV-light microscope that will fundamentally improve the success of our crystal screening campaigns and quality of our HQP training. The microscope would integrate into our X-ray crystallography facility at the University of Manitoba and fill a crucial technology gap that is needed to sustain our research and HQP training capabilities.