MACCHESS PROGRAM FOR MICROCRYSTALLOGRAPHY

微晶学 MACCHESS 程序

• 批准号: 8171505
• 负责人: RICHARD A. CERIONE
• 金额: $ 9万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8171505
• 关键词: Alzheimer' s Disease; Amyloid; Amyloid Fibrils; Benchmarking; Caliber; Cell Size; Cells; Complex; Computer Retrieval of Information on Scientific Projects Database; Confocal Microscopy; Data; Data Collection; Development; Dyes; Electronics; Encounter Groups; Exhibits; Fiber; France; Funding; G-Protein-Coupled Receptors; Grant; Harvest; Home environment; Image; Imagery; Institution; Ion Channel; Ireland; Mechanics; Medical; Membrane Proteins; Methodology; Methods; Mission; Needles; Non-Insulin-Dependent Diabetes Mellitus; Nosocomial Infections; Ohio; Optics; Peptides; Positioning Attribute; Prion Diseases; Property; Pseudomonas aeruginosa; Relative (related person); Research; Research Personnel; Resources; Robotics; Roentgen Rays; Sampling; Services; Source; System; Techniques; Technology; Testing; Texas; Three-Dimensional Imaging; Training; Tryptophan; United States National Institutes of Health; Work; base; beamline; biological systems; conformational conversion; detector; human disease; lens; light microscopy; pathogen; programs; protein aggregation; submicron; success

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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此，可以在其他清晰的条目中表示。列出的机构是 对于中心，这不一定是调查员的机构。 MacChess的微晶学术图极大地扩展了电台的能力，并显着提高了MacChess用户的成功样本的成功，正如成就中所示的。促进挑战生物系统的结构分析的方法，例如：1）复合骨料，例如构成与阿尔茨海默氏病相关的淀粉样蛋白纤维（Eisenberg，UCLA，UCLA）[102]，2）膜蛋白，尤其是这些膜蛋白，尤其是这些膜蛋白，尤其是那些蛋白，尤其是这些复合物聚集。与铜绿假单胞菌相关，铜绿假单胞菌是导致许多医院获得感染的机会性病原体（Caffrey，Limerick，Ireland和Ohio State Univ。 ）和生物医学上重要的G蛋白偶联受体（美国德克萨斯州纳瓦罗（Navarro））。以下是这些合作者面临的挑战的简要摘要，这些挑战激发了微晶技术计划。有关合作者工作的更多信息，请参见第D2.2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病，可传染的海绵脑病和II型糖尿病。尽管艾森伯格组倾向于形成纤维而不是常规的晶格，但艾森伯格集团还是成功地产生了关键淀粉样蛋白形成肽的微晶。这些超小针的针通常在窄直径中为1微米，需要特殊的收获和安装技术。 迄今为止，仅使用法国格勒诺布尔的ESRF的微晶光束线才能获得可用的衍射数据，这是美国研究人员通常不可用的设施。这些原纤维晶体应对用于在梁线上定位的光学显微镜的极限。它们是样品干式安装的独特例子，它们的小度是样品定位机械精度的重要基准。 X射线照明体积的较小与晶体的相对耐用性及其小单元相结合，可为所提出的微型CCD检测器产生出色的测试用例（如下所述）。原纤维也表现出高度可变的质量，因此必须筛选多个样品以获取最佳数据。 由Mackinnon组生长的具有挑战性的膜蛋白晶体通常也很小（<20微米），其衍射质量往往是可变的。可变性有时可能意味着几个晶体适合数据收集。因此，Mackinnon博士鼓励我们开发方法，以优化小晶体上的数据收集，并实施机器人技术以迅速筛选大量数字以识别有用的晶体。由Caffrey和Navarro组生长的膜相关蛋白质晶体提出了其他挑战。除了它们很小，脆弱且具有显着的单位细胞大小的事实外，晶体生长的不寻常矩阵（例如立方脂质中间的中间酶），提出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（例如共聚焦显微镜）应该有价值。我们建议探索如何使用微束，样品操纵和高级可视化方法的组合来识别其他有缺陷晶体的优质区域。 在这方面，康奈尔（Cornell）是世界上一个多光共聚焦显微镜中心之一的所在地。生物物理成像光电学（DRBIO）的发展资源目前正在开发其共聚焦显微镜技术的腹腔镜版本，该版本具有与光束线使用所需的相似的外形和光学要求。我们建议利用DRBIO专业知识（Warren Zipfel教授）来研究适应我们当前的光学元件或使用廉价的非球形透镜系统来实现基于天然（色氨酸）或染色诱导的粉料的亚光子体3D成像晶体样品的可行性。 所有四个协作组都遇到了许多样本不均匀性和晶体缺陷的情况。我们建议还与各种用户一起使用微束，作为MacChess服务，培训和传播任务的一部分，以检查水晶质量，以帮助制定策略来定位大部分水晶，并帮助用户获得获得的策略有用的数据。