Protein Beam Diffraction

该奖项为一个项目提供了持续的支持，该项目的最终目标是开发新的仪器，用于确定不能结晶的蛋白质和蛋白质复合物的三维原子结构。初步工作已通过先前的NSF探索性研究小额赠款（SGER）的PI的支持。 拟议的仪器将产生一束冻结的水合分子，该分子穿过同步加速器X射线束以产生用于结构测定的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物的液滴组成;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涂层。液滴在通过X射线束之前将与偏振红外激光对准。 将连续收集一个取向的所得散射图案，直到获得足够的数据，然后改变激光偏振的方向，并获得新的图案。这个过程将重复进行，直到有足够的数据从多个方向创建一个断层图像的蛋白质的电荷密度分布。椭圆偏振激光的使用允许所有分子轴的方向（但不是方向）对齐。相位问题将通过基于Gerchberg-Saxton-Feinup算法的迭代方法来解决。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逐渐减小分子的大小。挑战包括液滴对准的准确性、阻尼时间和反冲等问题。该项目使用跨学科研究团队开发的仪器和方法来解决一个公认重要的问题。这个建议的智力价值在于它对现代生物学中一个非常重要的问题的高度原创性的方法。如果成功的话，该项目将能够更好地理解现有结构方法难以处理的蛋白质类折叠的机制和能量景观。此外，通过避免结晶的需要，它可以大大提高所有类型蛋白质的结构测定速率。

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.