MACCHESS PROGRAM FOR AUTOMATION AND HIGH-THROUGHPUT

用于自动化和高吞吐量的 MACCHESS 程序

• 批准号: 7955572
• 负责人: EDWARD ARNOLD
• 金额: $ 4.15万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7955572
• 关键词: Apoptosis; Automation; Binding; Biological; Caspase; Categories; Cells; Communities; Complement; Computer Retrieval of Information on Scientific Projects Database; Computer software; Computers; Computing Methodologies; Crystallization; Crystallography; Data; Development; Funding; Goals; Grant; HIV; Institution; Laboratories; Lead; Methodology; Methods; Mind; Molecular Structure; Optics; Peptide Hydrolases; Performance; Phase; Population; Procedures; RNA Polymerase II; RNA-Directed DNA Polymerase; Research; Research Personnel; Resources; Robotics; Roentgen Rays; Scientist; Screening procedure; Signaling Protein; Sister; Site; Software Tools; Source; Speed; Structure; Synchrotrons; Technology; Time; United States National Institutes of Health; Xenon; base; beamline; data acquisition; design; detector; improved; interest; method development; new technology; pressure; programs; research and development; research study; software development; structural genomics; tool

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. "High-throughput" refers to procedures to increase the number of crystals analyzed per unit beam time. This involves a broad category of approaches, including automation for crystallization, handling, and diffraction, computer methods for faster data acquisition and analysis, and better X-ray detectors. Continued development of methods to increase crystallographic throughput is important for all synchrotron crystallographic resources. The overall needs of the crystallographic community are diverse and will involve, for example, fully-robotic beamlines designed to quickly obtain relatively lowresolution data from crystals with small- to mid-sized unit cells for structural genomics. The development of such beamlines is highly appropriate for the larger DOE sources. However, because CHESS is a small resource with a very limited number of stations, it is necessary to carefully choose highthroughput projects that complement and strengthen other proposed MacCHESS technological developments. Robotics-based technology is being intensively developed at our sister DOE-based sources. Mac- CHESS will continue to collaborate with other synchrotron sources to implement and adapt the technology that is developed. Thus, for example, we have implemented technology developed at the ALS for automatic mounting of crystals onto the goniometer of the beamline diffractometer. This capability will allow the rapid screening of microcrystals discussed in section D.1.1. However, we do not intend to devote a station to full-time automated crystallography because this is incompatible with the strength of MacCHESS, which has been a close interaction between the users, collaborators and MacCHESS scientists to develop new technology by application to challenging projects. Rather, the proposed MacCHESS high-throughput projects concentrate on improving aspects of the crystallographic experiment (e.g., robotics, new phasing procedures, detectors, and computer tools) that would have the overall effect of speeding acquisition and analysis of macromolecular structures. With these goals in mind, MacCHESS and collaborators will develop the necessary methodology to fulfill the need to screen and analyze large numbers of crystals of biological interest. These projects will include structural studies of the reverse transcriptase of HIV (Arnold laboratory, Rutgers U.), the analysis of signaling proteins and proteases (caspases) that lead to programmed cell death (Shi laboratory, Princeton U.), and efforts to determine X-ray structures for RNA polymerase II and associated binding partners (Fu laboratory, Cornell). Each of these efforts has great difficulty identifying diffraction- quality crystals among large crystal populations. Phasing Data acquisition times are now so short and computational hardware performance sufficiently powerful to realistically allow the synchrotron experimenter to perform on-site phasing and refinement. MAD/SAD phasing is an important tool for macromolecular crystallographers and our goal is to enable our collaborators and users to obtain phases within minutes after data reduction in most cases. Most commonly used software such as CCP4, CNS, SOLVE, XDS and SnB are already available at the beamline computers. In particular, as the SAPI, ABS and OASIS programs within the CCP4 suite are primarily developed at MacCHESS, users will benefit from the most up-to-date advances in methods development. We propose to continue the development of software tools for rapid phasing. We will also progressively extend MAD/SAD phasing to more challenging structures. Collaborative and core research in MAD/SAD phasing will benefit from several technical R&D projects involving low bandpass optics, software development and derivatization methods such as high pressure xenon introduction followed by pressure-cryocooling.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 中心，不一定是研究者的机构。 “高通量”是指增加每单位光束时间分析的晶体数量的程序。这涉及广泛的方法，包括结晶、处理和衍射的自动化、用于更快数据采集和分析的计算机方法以及更好的 X 射线探测器。 持续开发提高晶体学通量的方法对于所有同步加速器晶体学资源都很重要。晶体学界的总体需求是多种多样的，例如，将涉及全机器人光束线，旨在从具有中小型晶胞的晶体中快速获取分辨率相对较低的数据，用于结构基因组学。这种光束线的开发非常适合美国能源部较大的光源。然而，由于 CHESS 是一种小型资源，站点数量非常有限，因此有必要仔细选择高吞吐量项目，以补充和加强其他拟议的 MacCHESS 技术开发。 基于机器人技术的技术正在我们位于美国能源部的姐妹机构中得到集中开发。 MacCHESS 将继续与其他同步加速器源合作，实施和调整已开发的技术。例如，我们已经实施了 ALS 开发的技术，用于将晶体自动安装到光束线衍射仪的测角仪上。此功能将允许快速筛选 D.1.1 节中讨论的微晶。然而，我们并不打算将一个站专门用于全职自动化晶体学，因为这与 MacCHESS 的优势不相容，MacCHESS 一直是用户、合作者和 MacCHESS 科学家之间的密切互动，通过应用于具有挑战性的项目来开发新技术。相反，拟议的 MacCHESS 高通量项目集中于改进晶体学实验的各个方面（例如机器人技术、新的定相程序、探测器和计算机工具），这将产生加速大分子结构采集和分析的总体效果。考虑到这些目标，MacCHESS 和合作者将开发必要的方法来满足筛选和分析大量具有生物学意义的晶体的需求。这些项目将包括 HIV 逆转录酶的结构研究（罗格斯大学阿诺德实验室）、导致程序性细胞死亡的信号蛋白和蛋白酶（半胱天冬酶）的分析（普林斯顿大学施实验室），以及确定RNA 聚合酶 II 和相关结合伴侣的 X 射线结构（Fu 实验室，康奈尔大学）。这些努力中的每一项都很难在大型晶体群中识别衍射质量的晶体。 定相数据采集时间现在非常短，计算硬件性能足够强大，可以让同步加速器实验者实际执行现场定相和细化。 MAD/SAD 定相是大分子晶体学家的重要工具，我们的目标是使我们的合作者和用户在大多数情况下能够在数据缩减后几分钟内获得相。最常用的软件如 CCP4、CNS、SOLVE、XDS 和 SnB 已在光束线计算机上提供。特别是，由于 CCP4 套件中的 SAPI、ABS 和 OASIS 程序主要在 MacCHESS 上开发，因此用户将受益于方法开发的最新进展。我们建议继续开发软件工具以实现快速分阶段。 我们还将逐步将 MAD/SAD 定相扩展到更具挑战性的结构。 MAD/SAD 定相的合作和核心研究将受益于涉及低带通光学、软件开发和衍生化方法（例如高压氙引入和压力低温冷却）的多个技术研发项目。