Protein Beam Diffraction

该奖项为一个项目提供持续支持，该项目的最终目标是开发新的仪器，用于确定蛋白质和蛋白质复合体的三维原子结构，这些蛋白质和蛋白质复合体不能结晶。前期工作得到了美国国家科学基金会先前向PI提供的探索性研究小额补助金(SGER)的支持。拟议的仪器将产生一束冷冻的水合分子，它穿过同步加速器X射线束，产生用于结构确定的X射线散射图案。分子束将由含有一个蛋白质分子或大分子复合体的液滴组成；每个液滴在进入真空室时都会冻结，在分子周围形成一层薄薄的玻璃状冰层。在通过X射线束之前，液滴将与偏振红外激光对准。连续收集一个方向的散射图，直到获得足够的数据，然后改变激光偏振方向，获得新的图样。这个过程将被重复，直到有足够的来自多个方向的数据来创建蛋白质的电荷密度分布的断层图像。椭圆偏振激光的使用允许所有分子轴的方向(但不是感觉)对准。相位问题将采用基于Gerchberg-Saxton-Feinup算法的迭代方法来求解。最初的努力将使用烟草花叶病毒作为模型系统，然后在较低的温度和较高的激光功率下进行分子尺寸减小的研究。挑战包括液滴对准的准确性、减震时间和反冲等问题。该项目利用一个跨学科研究小组将要开发的仪器和方法，解决了一个公认的重要问题。这一提议的学术价值在于它对现代生物学中一个非常重要的问题采取了非常独到的方法。如果成功，该项目将使人们能够更好地理解蛋白质折叠的机制和能量格局，这些蛋白质的结构对现有的结构方法一直难以实现。此外，通过避免结晶的需要，它可以大大提高所有类型的蛋白质的结构确定的速度。

This award provides continued support for a project whose ultimate aim is the development of new instrumentation for determination of the three-dimensional atomic structure of protein and protein complexes that cannot be crystallized. Preliminary work has been supported through a prior NSF Small Grant for Exploratory Research (SGER) to the PIs. The proposed instrumentation will produce a beam of frozen hydrated molecules that traverses a synchrotron X-ray beam to produce an X-ray scattering pattern to be used for structure determination. The molecular beam will consist of droplets that contain one protein molecule or macromolecular complex; each droplet will freeze as it enters a vacuum chamber, forming a thin vitreous coating of ice around the molecule. Droplets will be aligned with a polarized infrared laser before passing through the X-ray beam. The resulting scattering pattern for one orientation will be collected continuously until sufficient data is acquired, then the direction of laser polarization will be changed, and a new pattern acquired. This process will be repeated until there is sufficient data from multiple orientations to create a tomographic image of the protein's charge-density distribution. The use of an elliptically polarized laser permits alignment of the direction (but not the sense) of all molecular axes. The phase problem will be solved by iterative methods based on the Gerchberg-Saxton-Feinup algorithm. Initial efforts will use tobacco mosaic virus as a model system, then progress to molecules of decreasing size at lower temperatures and higher laser power. Challenges include issues such as the accuracy of droplet alignment, damping times and recoil. The project addresses a problem of recognized importance using instrumentation and methodology to be developed by an interdisciplinary team of researchers. The intellectual merit of the proposal lies in its highly original approach to a problem of the great importance in modern biology. If successful, the project will enable improved understanding of the mechanisms and energetic landscape for the folding of classes of proteins whose structure has been refractory to existing structural approaches. Furthermore, by avoiding the need for crystallization, it may substantially increase the rate of structure determination for proteins of all types.