Crystallography without crystals: Atomic structure determination of laser oriente

描述(由申请人提供)：本申请涉及广泛的挑战领域(06)使能技术和具体的挑战主题，06-GM-101：大分子结构分析。我们计划开发一种新的方法，从定向的单分子的电子散射来确定蛋白质的原子结构，从而消除结晶学对单晶的依赖。在这个项目中，从电喷雾电离源产生的蛋白质离子将被嵌入到超流氦液滴的脉冲流中。含有离子的液滴光束将与椭圆偏振激光光束和相干高能电子束垂直相交。激光束是用来控制离子取向的。对离子不同取向下的连续电子衍射图进行过采样，为原子结构的确定提供了足够的信息。超流氦液滴束的极低温度低至0.38K，这对激光诱导的有效取向和高分辨电子衍射是非常有利的。我们的计划是在两年内证明这一想法的可行性。在第一年，我们将实现嵌入在超流氦液滴中的天然或接近天然的蛋白质离子的定向。我们将首先遵循有记录的蛋白质离子源设计，以产生高通量的天然或接近天然蛋白质离子。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/离子拦截区域下游增加一个检测室，并使用激光从少数荧光蛋白质中诱导荧光。这一步是为了确认嵌入离子的构象。在将定向激光引入检测室后，我们可以使用荧光蛋白质的线性二色性光谱来测量取向度。同时，我们将对现有的用于脉冲电子衍射的透射式电子显微镜进行改造。然后，我们可以组装完整的实验仪器，并使用几个标准蛋白质进行“概念验证”的实验。这一步骤还包括使用相检索和结构精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为该项目及时取得成功作出了贡献。首先，Pi(孔氏)研究小组是世界上唯一专攻场诱导取向和超流氦液滴冷却的小组，从理论建模到实验观察。其次，从连续衍射图的过采样恢复位相的理论原理和实验证明启发了单分子衍射领域，世界各地对超快X射线自由电子激光设施进行了重大投资。第三，质谱界在产生用于二级和高级结构研究的近天然蛋白质方面取得了长足的进步。通过结合这些来自不同领域的技术突破，我们希望成功地最终征服没有晶体的结晶学。这个项目有可能改变结晶学的范式。虽然这一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超快或连续的X射线源。最终，这种类型的用户设施可以建立在光束线上，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的占空比和辐射源的占空比，样品消耗可以减少到毫微摩尔，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、蛋白质复合体和纳米材料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。由于节省了大量的人力、时间和金钱，关于疾病机制和激进治疗策略的大胆假设可以及时和经济有效地从结构信息中得到测试。一个想法将不再仅仅因为一个人不能证明为了结构证据而需要投入大量时间和精力来生长足够大尺寸的单晶而被拒绝。因此，减少或取消结晶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。 公共卫生相关性：如果成功，该项目有可能极大地加快癌症、艾滋病和糖尿病等一系列疾病的机械性和治疗性研究的速度。药学研究中的大多数药物靶标都涉及难以或不可能结晶的蛋白质，该项目将消除结晶学对单晶的依赖。由于节省了大量的人力、时间和金钱，关于疾病机制和激进治疗策略的大胆假设可以及时和经济有效地从结构信息中得到测试。

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.