Crystallography without crystals: Atomic structure determination of laser oriente

描述（由申请人提供）：该申请涉及广泛的挑战领域（06）使能技术和特定的挑战主题，06- gm -101：大分子复合物的结构分析。我们计划开发一种新的方法，通过定向单分子的电子散射来确定蛋白质的原子结构，从而消除晶体学对单晶的依赖。在这个项目中，由电喷雾电离源产生的蛋白质离子将嵌入超流氦液滴的脉冲流中。含离子的液滴束与椭圆偏振激光束和相干高能电子束正交相交。激光束的作用是控制离子的方向。对离子不同取向下的连续电子衍射图进行过采样，为确定原子结构提供了充分的信息。超流氦滴光束的极低温度（低至0.38 K）对有效的激光诱导取向和高分辨率电子衍射极为有利。我们的计划是在两年内证明这个想法的可行性。在第一年，我们将实现嵌入超流氦滴的天然或接近天然蛋白质离子的定向。我们将首先遵循蛋白质离子源的文件设计，以产生高通量的天然或接近天然的蛋白质离子。在加入超流氦液滴源拦截蛋白质离子进行有效冷却后，我们将在液滴/离子拦截区下游增加一个检测室，并使用激光诱导少数荧光蛋白发出荧光。这一步是确认嵌入离子的构象。在检测室内引入定向激光后，我们可以使用荧光蛋白的线性二色光谱来测量定向程度。同时，我们将改进现有的透射电子显微镜用于脉冲电子衍射。然后，我们可以组装完整的实验装置，并使用一些标准蛋白质进行“概念验证”实验。这一步还包括使用相位检索和结构优化软件来获得蛋白质的原子结构，并将结果与蛋白质数据库中的可用信息进行比较。过去十年的几项关键技术发展有助于该项目的及时成功。首先，从理论建模到实验观察，PI （Kong）课课组是世界上唯一一个专门研究场诱导定向和超流氦滴冷却的课课组。其次，连续衍射图的过采样相位恢复的理论原理和实验证明激发了单分子衍射领域的发展，世界各地都在超快x射线自由电子激光设备上进行了大量投资。第三，质谱界在生成近天然蛋白用于二级和高阶结构研究方面取得了长足的进步。通过结合这些不同领域的技术突破，我们希望能够成功地最终征服无晶体的晶体学。这个项目有可能改变晶体学的范式。虽然这个阶段的辐射源是脉冲相干电子束，但基本的工作原理同样适用于x射线源，无论是超快的还是连续的。最终，这种类型的用户设施可以建立在光束线，如斯坦福直线加速器中心或国家同步加速器光源II。通过匹配电喷雾电离源与辐射源的占空比，样品消耗可以减少到飞摩尔，即使是最难表达的蛋白质也可以实现这一目标。随着大分子、蛋白质复合物和纳米材料的喷涂技术的进一步发展，晶体生长的困难和不可预测的过程将不再是强制性的。由于节省了大量人力、时间和金钱，对疾病机制和根治性治疗策略的大胆假设可以从结构信息中以及时和经济有效的方式进行测试。一个想法将不再仅仅因为无法证明需要投入大量时间和精力来培育足够大尺寸的单晶以作为结构证据而被驳回。因此，降低或消除晶体学的尺寸限制可以给生物科学家的思维方式带来根本性的转变。

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (06) Enabling Technologies and specific Challenge Topic, 06-GM-101: Structural analysis of macromolecular complexes. We plan to develop a new method for atomic structure determination of proteins from electron scattering of oriented single molecules, thereby eliminating the reliance of crystallography on single crystals. In this project, protein ions generated from an electrospray ionization source will be embedded in a pulsed stream of superfluid helium droplets. The droplet beam containing the ions is to orthogonally intercept an elliptically polarized laser beam and a coherent high energy electron beam. The laser beam is to control the orientation of the ions. Oversampling of the continuous electron diffraction patterns under different orientations of the ions offers sufficient information for atomic structural determination. The extreme low temperature of the superfluid helium droplet beam, down to 0.38 K, is extremely beneficial for effective laser induced orientation and high resolution electron diffraction. Our plan is to demonstrate the feasibility of this idea within two years. During the first year, we will achieve orientation of native or near native protein ions embedded in superfluid helium droplets. We will first follow a documented design of the protein ion source to produce a high flux of native or near native protein ions. After the addition of a superfluid helium droplet source to intercept the protein ions for effective cooling, we will then add a detection chamber downstream from the droplet/ion intercept region, and use a laser to induce fluorescence from a few fluorescing proteins. This step is to confirm the conformation of the embedded ion. After introducing an orientation laser into the detection chamber, we can use linear dichroism spectroscopy of the fluorescing protein to measure the degree of orientation. In the meantime, we will modify an existing transmission electron microscope for pulsed electron diffraction. We can then assemble the complete experimental apparatus and use a few standard proteins for the experiment of "proof of concept". This step also involves using the phase retrieval and structure refinement software to obtain the atomic structure of the protein and compare the result with the available information from the protein databank. Several key technological developments over the past decade contribute to the timely success of this project. First of all, the PI's (Kong's) research group is the only group in the world specializing in field induced orientation and superfluid helium droplet cooling, from theoretical modeling to experimental observation. Secondly, the theoretical principle and experimental demonstration of phase retrieval from oversampling of continuous diffraction patterns have inspired the field of single molecule diffraction, with major investments in ultrafast x-ray free electron laser facilities throughout the world. Thirdly, the mass spectrometry community has taken great strides in generating near native proteins for secondary and higher order structure studies. By combining these technological breakthroughs from different fields, we hope to succeed in the ultimate conquest of crystallography without crystals. This project has the potential for shifting the paradigm of crystallography. Although the radiation source at this stage is pulsed coherent electron beams, the fundamental principle of operation is equally applicable to x-ray sources, either ultrafast or continuous. Ultimately, user facilities of this type can be established at beamlines such as the Stanford Linear Accelerator Center or National Synchrotron Light Source II. By matching the duty cycle of an electrospray ionization source with that of the radiation source, sample consumption can be reduced to femtomoles, a target achievable even for the most difficult protein to express. With further development in the spraying technology for macromolecules, protein complexes, and nanomaterials, the difficult and yet unpredictable process of crystal growth will no longer be mandatory. With tremendous savings in human effort, time, and money, daring hypotheses on disease mechanisms and radical therapeutic strategies could be tested from structural information in a timely and cost-effective manner. An idea would no longer have to be dismissed simply because one cannot justify a significant investment of time and effort needed to grow a sufficiently large sized single crystal for structural evidence. Decreasing or eliminating the size limit for crystallography can thus bring a fundamental transformation in the mindset of biological scientists. PUBLIC HEALTH RELEVANCE: If successful, this project has the potential to dramatically accelerate the rate of mechanistic and therapeutic studies for a gamut of diseases, including cancer, AIDS and diabetes. Most drug targets in pharmaceutical research involve proteins that are difficult or impossible to crystallize, and this project will eliminate the reliance of crystallography on single crystals. With tremendous savings in human effort, time, and money, daring hypotheses on disease mechanisms and radical therapeutic strategies could be tested from structural information in a timely and cost-effective manner.