Diffraction From Microvolume Topaz Microprocessors

描述（由申请人提供）：我们打算开发一种改进的微流体装置，该装置可以直接用x射线辐射进行结晶实验，而无需从井中取出晶体。这将使实验人员能够根据衍射数据而不是任意的光学判断来评估结晶实验的结果。鉴定生长条件的x射线衍射质量晶体仍然是一个限制在大分子晶体学。这是一个特殊的问题，在样品的情况下，只能在有限的数量制备，如膜蛋白和多组分大分子复合物。Fluidigm公司已经开发并商业化了TOPAZ(r)系统，该系统使用微流控芯片进行结晶，每次实验的样品体积最小（<10nl）。我们将开发的新芯片产品将与TOPAZ结晶系统完全兼容。该芯片将被设计为大批量生产，并以最终用户合理的成本。在第一阶段，我们设计了微流控芯片，可以在室温和低温下收集衍射数据。第一阶段的实验证明了从结晶实验中原位收集衍射数据的能力，可以快速确定1)晶体是蛋白质还是盐，以及2)晶体的衍射质量。在第二阶段，除了开发芯片外，我们打算修改劳伦斯伯克利国家实验室（LBNL）先进光源（ALS）的光束线8.3.1和12.3.1端站。测角仪将被修改，以容纳TOPAZ载体和定位井，以供x射线束审问。还将开发适当的软件来自动控制芯片在光束线上的定位和对准。TOPAZ测角仪将被设计成与其他x射线衍射仪兼容。我们已经证明，含有晶体的黄玉芯片可以在室温下运输，对晶体几乎没有明显的损害。我们将用合作实验室的样品来表征这种传输行为，并在LBNL研究基于衍射的芯片筛选服务的发展。Fluidigm和LBNL的技术和科学优势相结合，将能够开发出一种强大的、可商用的产品，适用于基于衍射的大分子晶体筛选。这不仅将大大增强现有的晶体学方法，而且还将提高成功确定结构的率。7. 项目说明：蛋白质和核酸结构的测定对人类疾病的理解和治疗产生了深远的影响。该项目将开发一种商业产品，消除确定这些结构的重大瓶颈-在确定生长条件后立即评估用于衍射研究的晶体的质量。此外，这将在微流控芯片中进行，使用微小体积的样品，并且在数据收集之前不需要触摸或处理晶体。

DESCRIPTION (provided by applicant): We intend to develop an improved microfluidic device that allows crystallization experiments to be interrogated directly with X-ray radiation without requiring crystals to be removed from the well. This will allow experimenters to assess the outcome of crystallization experiments on the basis of diffraction data rather than arbitrary optical judgments. Identification of conditions for growth of X-ray diffraction quality crystals continues to be a limitation in macromolecular crystallography. This is a particular problem in the case of samples that can only be prepared in limited quantities, such as membrane proteins and multi-component macromolecular complexes. Fluidigm Corp. has developed and commercialized the TOPAZ(r) system, that uses microfluidic chips for crystallization using minimal (<10nl) sample volumes per experiment. The new chip product we will develop will be fully compatible with the TOPAZ crystallization system. The chip will be designed to be manufactured in large quantities, and with a cost that is reasonable for the end user. During Phase I, we designed microfluidic chips from which diffraction data could be collected at both ambient and cryogenic temperatures. Phase I experiments demonstrated the ability to collect diffraction data from crystallization experiments in situ, allowing rapid determination of 1) whether crystals are protein or salt, and 2) the diffraction quality of the crystal. During phase II, in addition to development of the chip, we intend to modify the end stations of Beamlines 8.3.1 and 12.3.1 at the Advanced Light Source (ALS) at Lawrence Berkeley National Laboratory (LBNL). The goniometers will be modified to hold the TOPAZ carrier and position wells for interrogation by the X-ray beam. Appropriate software will also be developed to automate control of the positioning and alignment of the chip at the beamline. The TOPAZ goniometer will be designed to be compatible with other X-ray diffractometers. We have shown that TOPAZ chips containing crystals can be transported at room temperature with little apparent damage to the crystals. We will characterize this transport behavior with samples from collaborator labs, and investigate the development of a diffraction-based chip screening service at LBNL. The combination of technical and scientific strengths at Fluidigm and LBNL will enable development of a robust, commercially-available product suitable for diffraction-based screening of macromolecular crystals. This will provide not only a significant enhancement to currently available crystallographic methods, but also an increase in the rate of successful structure determination. 7. Project Narrative: Determination of protein and nucleic acid structures has had a profound influence on understanding and developing treaments for human disease. This project will develop a commercial product that will eliminate a significant bottleneck in determination of these structures-assessing the quality of crystals used for diffraction studies immediately after the conditions to grow them have been identified. Moreover, this will be carried out in a microfluidic chip, using tiny volumes of sample, and without requiring the crystals ever to be touched or handled before data collection.