MACCHESS PROGRAM FOR MICROCRYSTALLOGRAPHY
微晶学 MACCHESS 程序
基本信息
- 批准号:8363525
- 负责人:
- 金额:$ 1.14万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2011
- 资助国家:美国
- 起止时间:2011-07-01 至 2012-06-30
- 项目状态:已结题
- 来源:
- 关键词:Alzheimer&aposs DiseaseAmyloidAmyloid FibrilsBenchmarkingCaliberCell SizeCellsComplexConfocal MicroscopyDataData CollectionDevelopmentDyesElectronicsEncounter GroupsExhibitsFiberFranceFundingG-Protein-Coupled ReceptorsGrantHarvestHome environmentImageImageryIon ChannelIrelandMechanicsMedicalMembrane ProteinsMethodologyMethodsMissionNational Center for Research ResourcesNeedlesNon-Insulin-Dependent Diabetes MellitusNosocomial InfectionsOhioOpticsPeptidesPositioning AttributePrincipal InvestigatorPrion DiseasesPropertyPseudomonas aeruginosaRelative (related person)ResearchResearch InfrastructureResearch PersonnelResourcesRoboticsRoentgen RaysSamplingServicesSourceSystemTechniquesTechnologyTestingTexasThree-Dimensional ImagingTrainingTryptophanUnited States National Institutes of HealthWorkbasebeamlinebiological systemsconformational conversioncostdetectorhuman diseaselenslight microscopypathogenprogramsprotein aggregationsubmicronsuccess
项目摘要
This subproject is one of many research subprojects utilizing the resources
provided by a Center grant funded by NIH/NCRR. Primary support for the subproject
and the subproject's principal investigator may have been provided by other sources,
including other NIH sources. The Total Cost listed for the subproject likely
represents the estimated amount of Center infrastructure utilized by the subproject,
not direct funding provided by the NCRR grant to the subproject or subproject staff.
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.
该子项目是利用资源的众多研究子项目之一
由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持
并且子项目的主要研究者可能是由其他来源提供的,
包括其他 NIH 来源。 子项目可能列出的总成本
代表子项目使用的中心基础设施的估计数量,
NCRR 赠款不直接向子项目或子项目工作人员提供资金。
MacCHESS 的微晶体学极大地扩展了工作站的能力,并显着提高了 MacCHESS 用户处理困难样品的成功率,正如 C 部分的成就中所说明的那样。在接下来的项目期间,我们将与主要合作者密切合作,进一步开发微晶体促进具有挑战性的生物系统的结构分析的方法,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聚集体(Eisenberg,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,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(爱尔兰利默里克大学卡弗里和俄亥俄州立大学),3)门控离子通道功能所需的特性和构象转变(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,美国德克萨斯州医学分会)。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的发展。有关合作者工作的更多信息请参见 D.2.2 节。 许多重要的人类疾病都与蛋白质的有害聚集有关。最著名的是阿尔茨海默病、传染性海绵状脑病和 II 型糖尿病。艾森伯格小组已成功生产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。这些超小针的直径通常为 1 微米,需要特殊的采集和安装技术。 迄今为止,可用的衍射数据只能通过法国格勒诺布尔 ESRF 的微晶体学光束线获得,而美国研究人员通常无法使用该设施。这些原纤维晶体突破了用于光束线定位的光学显微镜的极限。它们是样品干安装的独特示例,其体积小是样品定位机械精度的重要基准。 X 射线照射体积较小,与晶体及其小晶胞的相对耐用性相结合,为所提出的微型 CCD 探测器(如下所述)提供了出色的测试用例。原纤维还表现出高度可变的质量,因此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存在差异。变异性有时可能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,并使用机器人技术来快速筛选大量晶体以识别有用的晶体。卡弗里和纳瓦罗小组培育的膜相关蛋白晶体带来了额外的挑战。除了它们体积小、脆弱且晶胞尺寸大之外,晶体生长的不寻常基质(例如立方脂质中间相)也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(例如共焦显微镜)应该是有价值的。我们建议探索如何结合使用微束、样品操作和先进的可视化方法来识别有缺陷的晶体上的优质区域。 在这方面,康奈尔大学是世界多光子共焦显微镜中心之一的所在地。生物物理成像光电发展资源 (DRBIO) 目前正在开发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外形尺寸和光学要求。我们建议利用 DRBIO 的专业知识(Warren Zipfel 教授)来研究采用我们当前的光学器件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,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。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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RICHARD A. CERIONE其他文献
RICHARD A. CERIONE的其他文献
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Targeting the dependency of cancer cells on the sirtuin SIRT5
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Targeting the dependency of cancer cells on the sirtuin SIRT5
靶向癌细胞对 Sirtuin SIRT5 的依赖性
- 批准号:
10261077 - 财政年份:2019
- 资助金额:
$ 1.14万 - 项目类别:
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