MACCHESS PROGRAM FOR AUTOMATION AND HIGH-THROUGHPUT

该子项目是利用 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”是指增加每单位射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射线检测器。 不断发展提高晶体学产量的方法对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样化的，例如，将涉及全自动光束线，旨在快速获得相对较低分辨率的数据，从晶体与小到中等大小的单位细胞的结构基因组学。这种光束线的发展非常适合于较大的DOE源。然而，由于国际象棋是一个小的资源与数量非常有限的站，有必要仔细选择高吞吐量的项目，补充和加强其他拟议的MacCHESS技术发展。 基于机器人的技术正在我们的姐妹能源部集中开发。Mac- CHESS将继续与其他同步加速器源合作，以实施和适应所开发的技术。因此，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。这种能力将允许快速筛选第D.1.1节中讨论的微晶。然而，我们不打算将一个站投入全职自动晶体学，因为这与MacCHESS的实力不相容，MacCHESS的优势是用户，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高通量项目集中在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），这将具有加速大分子结构的获取和分析的总体效果。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生物晶体的需求。这些项目将包括艾滋病毒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（Shi实验室，普林斯顿大学），以及确定RNA聚合酶II和相关结合配偶体的X射线结构的努力（Fu实验室，Cornell）。这些努力中的每一个都很难在大的晶体群中识别衍射质量的晶体。 相位数据采集时间现在如此之短，计算硬件性能足够强大，可以让同步加速器实验人员在现场进行相位调整和优化。 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能够在大多数情况下在数据简化后几分钟内获得相。最常用的软件，如CCP 4，CNS，SOLVE，XDS和SnB已经在光束线计算机上可用。特别是，由于CCP 4套件中的SAPI、ABS和OASIS程序主要是在MacCHESS开发的，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，以便快速分阶段进行。 我们还将逐步将MAD/SAD阶段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几个技术研发项目，涉及低通光学，软件开发和衍生方法，如高压氙气引入，然后压力低温冷却。

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.