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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 MacCHESS的微晶学极大地扩展了工作站的能力，并显著提高了MacCHESS用户处理困难样品的成功率，如C部分的成就所示。在未来的项目期间，我们将与主要合作伙伴密切合作，进一步发展微晶方法学，以促进具有挑战性的生物系统的结构分析，例如：1）复杂的聚集体，例如构成与阿尔茨海默病相关的淀粉样原纤维的那些（艾森伯格，加州大学洛杉矶分校）[102]，2）在脂质中间相中生长的膜蛋白，特别是与铜绿假单胞菌相关的膜蛋白，一种导致许多医院获得性感染的机会性病原体（Caffrey，利默里克大学，爱尔兰和俄亥俄州州立大学），3)离子通道功能所必需的门控性质和构象转变（MacKinnon，Rockefeller Univ.）和生物医学上重要的G蛋白偶联受体（Navarro，U. Texas Medical分支）。以下是这些合作者所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第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病，传染性海绵状脑病和II型糖尿病。艾森伯格小组已经成功地制造出了关键的淀粉样蛋白形成肽的微晶，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，可用的衍射数据只能使用法国格勒诺布尔ESRF的微晶光束线获得，这是一个美国研究人员不经常使用的设施。这些纤维晶体应变的限制，光学显微镜用于定位在光束线。它们是样品干镶嵌的独特例子，其微小性是样品定位机械精度的重要基准。X射线照射体积的小与晶体的相对耐久性及其小的单位晶胞相结合，为所提出的微型CCD检测器（如下所述）产生了极好的测试情况。原纤维还表现出高度可变的质量，使得有必要筛选多个样品以获得最佳数据。 MacKinnon小组生长的具有挑战性的膜蛋白晶体通常也很小（< 20微米），并且其衍射质量往往是可变的。这种可变性有时意味着只有百分之几的晶体适合数据收集。出于这个原因，麦金农博士鼓励我们开发方法来优化小晶体的数据收集，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、易碎和具有显著晶胞尺寸的事实之外，晶体生长的不寻常基质（例如立方晶系中间相）呈现出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，应该证明是有价值的。我们建议探索如何结合微束，样品操作和先进的可视化方法可以用来识别质量好的区域，否则有缺陷的晶体。 在这方面，康奈尔大学是世界多光子共聚焦显微镜中心之一。生物物理成像光电发展资源（DRBIO）目前正在开发其共焦显微镜技术的腹腔镜版本，该技术具有与光束线使用所需的相似的形状因子和光学要求。我们建议利用DRBIO专业知识（Warren Zipfel教授）来研究适应我们当前光学器件或使用廉价的非球面透镜系统的可行性，以实现基于天然（色氨酸）或染料诱导荧光的亚微米3D成像晶体样品。 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。我们还建议与广大用户合作，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。