MACCHESS PROGRAM FOR MICROCRYSTALLOGRAPHY

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 MacCHESS的微晶学极大地扩展了空间站的能力，并大大增加了使用困难样本的MacCHESS用户的成功，正如在C节的成就中所表明的那样。在接下来的项目期，我们将与主要合作者密切合作，进一步开发微晶方法学，以促进对具有挑战性的生物系统的结构分析，如：1)复杂聚集体，如构成与阿尔茨海默病相关的淀粉样纤维的那些聚集体(Eisenberg，UCLA)[102]，2)在脂类中间相生长的膜蛋白，特别是与铜绿假单胞菌有关的那些微晶，铜绿假单胞菌是一种导致许多医院获得性感染的机会性病原体(Caffrey，Univ.利默里克，爱尔兰和俄亥俄州立大学)，3)离子通道功能所需的门控性质和构象转变(MacKinnon，洛克菲勒大学)和生物医学上重要的G蛋白偶联受体(纳瓦罗大学德克萨斯医学分部)。以下是这些合作者面临的挑战的简要总结，这些挑战推动了微晶技术计划。关于合作者工作的更多信息见D.2.2节。许多重要的人类疾病涉及蛋白质的有害聚集。最著名的是阿尔茨海默氏症、传染性海绵状脑病和II型糖尿病。艾森伯格团队已经成功地制造出关键的淀粉样蛋白形成肽的微晶体，尽管它们倾向于形成纤维，而不是规则的晶格。这些超小的针，通常是1微米的窄直径，需要特殊的收获和安装技术。到目前为止，只有使用法国格勒诺布尔ESRF的微晶学光束线才能获得可用的衍射数据，而美国研究人员并不经常使用这种设备。这些原纤状晶体使光学显微镜在光束线上定位的极限受到限制。它们是样品干式安装的独一无二的例子，它们的小巧是样品定位机械精度的重要基准。X射线照明体积小，再加上晶体及其小单元电池的相对耐用性，为建议的微型CCD探测器提供了极好的测试用例(如下所述)。原纤维还表现出高度可变的质量，因此有必要对多个样本进行筛选以获得最佳数据。由麦金农小组生长的具有挑战性的膜蛋白晶体通常也很小(20微米)，而且它们的衍射质量往往不同。这种可变性有时意味着，有百分之几的晶体适合收集数据。为此，麦金农博士鼓励我们开发方法，优化对小晶体的数据收集，并实施机器人技术来快速筛选大量晶体，以识别有用的晶体。由Caffrey和Navarro小组生长的膜相关蛋白质晶体带来了额外的挑战。除了它们小、易碎、单位晶胞大小很大这一事实外，生长晶体的不寻常基质(如立方脂类中间相)还带来了独特的可视化和收获挑战。在这种情况下，使用更复杂的可视化方法，如共聚焦显微镜，应该被证明是有价值的。我们建议探索如何结合微光束、样品处理和先进的可视化方法来识别其他有缺陷的晶体上的优质区域。在这方面，康奈尔大学是世界上多光子共焦显微镜中心之一的所在地。生物物理成像光电子学开发资源(DRBIO)目前正在开发他们的共焦显微镜技术的腹腔镜版本，该技术具有类似于光束线使用所需的形状因素和光学要求。我们建议利用DRBIO的专业知识(Warren Zipfel教授)来研究采用我们目前的光学系统或使用廉价的非球面透镜系统来实现基于天然(色氨酸)或染料诱导荧光的亚微米3D成像晶体样品的可行性。所有四个合作小组都遇到了许多样品不均匀和晶体缺陷的情况。我们还建议与广泛的用户合作使用微束，作为MacCHESS服务、培训和传播任务的一部分，以检查晶体质量，帮助制定定位晶体良好部分的策略，并帮助用户获得有用的数据。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Microcrystallography at MacCHESS greatly extends the capability of the stations and significantly increases the success of MacCHESS users with difficult samples, as has been illustrated in the accomplishments,Section C. In the coming project period, we will work closely with key collaborators to further develop microcrystal methodology to facilitate the structural analysis of challenging biological systems such as: 1) complex aggregates such as those that make up the amyloid fibrils associated with Alzheimer's disease (Eisenberg, UCLA) [102], 2) membrane proteins grown in lipidic mesophases, particularly those associated with Pseudomonas aeruginosa, an opportunistic pathogen responsible for many hospital-acquired infections (Caffrey, Univ. of Limerick, Ireland and Ohio State Univ.), 3) the gating properties and conformational transitions necessary for ion channel function (MacKinnon, Rockefeller Univ.) and biomedically important G protein-coupled receptors (Navarro, U. Texas Medical Branch). Below is a brief summary of the challenges confronted by these collaborators that motivate the microcrystal technical program. More information about the collaborators' work is given in section D.2.2. A number of important human diseases involve the harmful aggregation of proteins. Best known are Alzheimer`s disease, transmissible spongiform encephalopathies, and Type II diabetes mellitus. The Eisenberg group has managed to produce microcrystals of key amyloid-forming peptides, in spite of their tendency to form fibers rather than regular crystal lattices. These ultra-small needles, typically 1 micron in the narrow diameter, require special harvesting and mounting techniques. To date, usable diffraction data have only been obtainable using the microcrystallography beamline at the ESRF in Grenoble, France, a facility that is not often available to US researchers. These fibril crystals strain the limits of optical light microscopy used for positioning at beamlines. They are a unique example of sample dry mounting and their smallness serves as an important benchmark for mechanical precision of sample positioning. The smallness of X-ray illuminated volume combines with the relative durability of the crystals and their small unit cell to produce an excellent test case for the proposed micro CCD detectors (described below). Fibrils also exhibit highly variable quality, making it necessary to screen multiple samples to obtain optimal data. The challenging membrane protein crystals grown by the MacKinnon group are also often small (< 20 microns) and tend to be variable in their diffraction quality. The variability can sometimes mean that a few percent of the crystals are suitable for data collection. For this reason, Dr. MacKinnon had encouraged us to develop methods to optimize data collection on small crystals and to implement robotics to rapidly screen large numbers to identify useful crystals. Membrane associated protein crystals grown by the Caffrey and Navarro groups pose additional challenges. Beyond the fact that they are small, fragile, and of significant unit cell size, the unusual matrices in which the crystals are grown (such as cubic lipidic mesophases), present unique visualization and harvesting challenges. The use of more sophisticated visualization methods, such as confocal microscopy, should prove valuable in this case. We propose to explore how a combination of microbeams, sample manipulation and advanced visualization methods can be used to identify good quality regions on otherwise defective crystals. In this regard, Cornell is home to one of the world centers for multiphoton confocal microscopy. The Developmental Resource for Biophysical Imaging Opto-Electronics (DRBIO) is currently developing a laparoscopic version of their confocal microscopy technology which has similar form factor and optical requirements to what would be needed for beamline use. We propose to leverage DRBIO expertise (Prof. Warren Zipfel) to investigate the feasibility of either adapting our current optics or using an inexpensive aspherical lens system to achieve submicron 3D imaging crystal samples based on natural (tryptophan) or dye-induced flourescence. All four collaborating groups encounter many cases of sample inhomogeneity and crystal imperfection. We propose to also work with a wide range of our users in using microbeams, as part of the MacCHESS service, training, and dissemination missions, to examine crystal quality, to help develop strategies for locating good portions of crystal, and to help users obtain useful data.