Diffraction from Microvolume Topaz Microprocessors

描述（由申请人提供）：我们建议开发一种用于蛋白质晶体的有效和快速的X射线衍射筛选，这将加速难以表达和纯化的蛋白质的结构测定。在原子分辨率下确定蛋白质结构使我们对生物学的基本理解取得了重大进展，并为合理的药物设计提供了基础。然而，通过X射线晶体学确定蛋白质结构的主要障碍是在识别X射线衍射蛋白质晶体的初始步骤中。目前主要通过形态学然后通过衍射来识别蛋白质晶体的方法在时间和材料方面是昂贵的。我们将修改Fluidigm黄玉结晶芯片，使蛋白质晶体可以在同步加速器上进行原位X射线衍射筛选。我们将优化芯片的材料组成，以最大限度地提高X射线的透明度，同时保持结晶蛋白质的功能能力。然后，我们将开发区分蛋白质和污染盐晶体的参数，这些晶体小到10微米。我们将与多位结构生物学家合作，利用衍射屏幕与各种各样的跨膜，信号和酶蛋白。我们预计，我们将开发的衍射屏将很容易适应其他同步加速器设施，并将加速具有科学和医学重要性的蛋白质的晶体学研究。

DESCRIPTION (provided by applicant): We propose to develop an efficient and rapid X-ray diffraction screen for protein crystals that will accelerate structure determination of proteins that are difficult to express and purify. Determination of protein structures at atomic resolution has allowed major advances in our fundamental understanding of biology and has provided a basis for rational drug design. However, a major obstacle to determining protein structures by X-ray crystallography has been in the initial step of identifying X-ray diffracting protein crystals. Current methods that identify protein crystals primarily by morphology followed by diffraction are costly in terms of time and material. We will modify the Fluidigm Topaz crystallization chip so that protein crystals can be screened in situ for X-ray diffraction at a synchrotron. We will optimize the material composition of the chip to maximize X-ray transparency while maintaining the functional ability to crystallize proteins. We will then develop the parameters to differentiate protein and contaminating salt crystals that are as small as 10 microns. We will collaborate with multiple structural biologists to utilize the diffraction screen with a wide variety of transmembrane, signaling, and enzymatic proteins. We anticipate that the diffraction screen that we will develop will be easily adapted to other synchrotron facilities and will accelerate crystallographic studies of proteins with scientific and medical importance.