MACCHESS PROGRAM FOR AUTOMATION AND HIGH-THROUGHPUT

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 “高通量”指的是增加每单位光束时间分析的晶体数量的程序。这涉及一大类方法，包括结晶、处理和衍射的自动化，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。继续开发提高晶体产量的方法对所有同步加速器晶体资源都很重要。晶体学界的总体需求是多样的，例如，将涉及全自动光束线，旨在快速从具有中小型单位细胞的晶体中获得相对较低分辨率的数据，用于结构基因组学。这种波束线的发展对于较大的DOE源是非常合适的。然而，由于国际象棋是一种小资源，站的数量非常有限，因此有必要谨慎地选择高吞吐量项目，以补充和加强其他拟议的MacCHESS技术开发。基于机器人的技术正在我们以能源部为基础的姊妹工厂密集开发。Mac-Chess将继续与其他同步加速器来源合作，实施和适应所开发的技术。因此，例如，我们已经实施了在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。这一能力将允许快速筛选D.1.1节中讨论的微晶体。然而，我们不打算专门为全职自动化结晶学设立站点，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高通量项目专注于改进晶体实验的各个方面(例如，机器人技术、新的定相程序、探测器和计算机工具)，这些方面将产生加速获取和分析大分子结构的总体效果。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意义的晶体的需要。这些项目将包括艾滋病毒逆转录酶的结构研究(Arnold实验室，罗格斯大学)，导致细胞程序性死亡的信号蛋白和蛋白酶(Caspase)的分析(SHI实验室，普林斯顿大学)，以及确定RNA聚合酶II及其相关结合伙伴的X射线结构的努力(FU实验室，康奈尔大学)。这些努力中的每一项都很难在大的晶体群中识别出衍射质量的晶体。定相数据采集时间如此之短，计算硬件性能也足够强大，可以让同步加速器实验者在现场进行定相和精细化。MAD/SAD定相是大分子结晶学家的重要工具，我们的目标是使我们的合作者和用户在大多数情况下在数据简化后的几分钟内获得物相。最常用的软件，如CCP4、CNS、SOLUE、XDS和SNB已经可以在光束线计算机上使用。特别是，由于CCP4套件中的SAPI、ABS和OASIS计划主要是在MacCHESS开发的，用户将受益于方法开发方面的最新进展。我们建议继续开发用于快速分阶段的软件工具。我们还将逐步将MAD/SAD分阶段推广到更具挑战性的结构。MAD/SAD阶段化方面的协作和核心研究将受益于涉及低带通光学、软件开发和衍生方法的几个技术研发项目，例如高压氙气引入和压力低温冷却。

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.