Crystallography without crystals: Atomic structure determination of laser oriente

无晶体的晶体学：激光取向的原子结构测定

• 批准号: 7834012
• 负责人: Wei Kong
• 金额: $ 49.99万
• 依托单位: OREGON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7834012
• 关键词: Acquired Immunodeficiency Syndrome; Address; Algorithms; Area; Biological; Calibration; California; Communities; Computer software; Conquest; Consumption; Crystallography; Databases; Detection; Development; Diabetes Mellitus; Disease; Drug Delivery Systems; Electron Beam; Electron Microscope; Electrons; Electrospray Ionization; Fluorescence; Goals; Green Fluorescent Proteins; Growth; Guns; Helium; Higher Order Chromatin Structure; Human; Image; Intercept; Investments; Ions; Lasers; Light; Linear Accelerator Radiotherapy Systems; Los Angeles; Macromolecular Complexes; Malignant Neoplasms; Mass Spectrum Analysis; Measurement; Measures; Methods; Modification; Molecular Conformation; Motion; Myoglobin; Operative Surgical Procedures; Pattern; Pharmacologic Substance; Phase; Physiologic pulse; Process; Proteins; Radiation; Reliance; Research; Resolution; Rest; Retrieval; Risk; Sampling; Savings; Scientist; Source; Specimen; Spectrum Analysis; Staging; Stream; Structure; Synchrotrons; System; Techniques; Technology; Testing; Theoretical model; Therapeutic; Therapeutic Studies; Time; Universities; Vacuum; Work; base; beamline; biological research; cold temperature; cost; design; dichroism; experience; ion source; macromolecule; mass spectrometer; nanomaterials; protein complex; protein structure; public health relevance; research study; single molecule; software systems; success; transmission process; virtual; Acquired Immunodeficiency Syndrome; Address; Algorithms; Area; Biological; Calibration; California; Communities; Computer software; Conquest; Consumption; Crystallography; Databases; Detection; Development; Diabetes Mellitus; Disease; Drug Delivery Systems; Electron Beam; Electron Microscope; Electrons; Electrospray Ionization; Fluorescence; Goals; Green Fluorescent Proteins; Growth; Guns; Helium; Higher Order Chromatin Structure; Human; Image; Intercept; Investments; Ions; Lasers; Light; Linear Accelerator Radiotherapy Systems; Los Angeles; Macromolecular Complexes; Malignant Neoplasms; Mass Spectrum Analysis; Measurement; Measures; Methods; Modification; Molecular Conformation; Motion; Myoglobin; Operative Surgical Procedures; Pattern; Pharmacologic Substance; Phase; Physiologic pulse; Process; Proteins; Radiation; Reliance; Research; Resolution; Rest; Retrieval; Risk; Sampling; Savings; Scientist; Source; Specimen; Spectrum Analysis; Staging; Stream; Structure; Synchrotrons; System; Techniques; Technology; Testing; Theoretical model; Therapeutic; Therapeutic Studies; Time; Universities; Vacuum; Work; base; beamline; biological research; cold temperature; cost; design; dichroism; experience; ion source; macromolecule; mass spectrometer; nanomaterials; protein complex; protein structure; public health relevance; research study; single molecule; software systems; success; transmission process; virtual

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.

描述（由申请人提供）：此申请解决广泛的挑战领域（06）启用技术和特定挑战主题，06-gm-101：大分子复合物的结构分析。我们计划开发一种新方法来从定向单分子的电子散射中测定蛋白质的原子结构，从而消除了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到超氟氦液滴的脉冲流中。包含离子的液滴梁是正交截取椭圆极化激光束和连贯的高能电子束。激光束将控制离子的方向。离子不同方向下连续电子衍射模式的过采样提供了足够的原子结构测定信息。超氟氦液滴束的极端低温降至0.38 K，对有效激光诱导的方向和高分辨率电子衍射非常有益。我们的计划是在两年内证明这一想法的可行性。在第一年，我们将实现嵌入超氟氦液滴中的天然或靠近天然蛋白质离子的方向。我们将首先遵循蛋白质离子源的文献设计，以产生天然或靠近天然蛋白质离子的高通量。在添加了超氟氦液滴源以拦截蛋白质离子以进行有效冷却之后，我们将在液滴/离子截距区域下游添加一个检测室，并使用激光从几种荧光蛋白中诱导荧光。此步骤是确认嵌入离子的构象。将方向激光引入检测室后，我们可以使用荧光蛋白的线性二色性光谱来测量方向。同时，我们将修改现有的透射电子显微镜以进行脉冲电子衍射。然后，我们可以组装完整的实验设备，并使用一些标准蛋白进行“概念证明”的实验。此步骤还涉及使用相位检索和结构改进软件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。在过去十年中，几个关键的技术发展有助于该项目的及时成功。首先，PI（Kong's）研究小组是世界上唯一专门从事现场引起的方向和超流体氦液滴冷却的小组，从理论建模到实验性观察。其次，从连续衍射模式过度采样的相位检索的理论原理和实验证明激发了单分子衍射的领域，在全球范围内对超快X射线X射线游离电子激光设施进行了重大投资。第三，质谱界在生成近期和高阶结构研究附近的天然蛋白质方面取得了长足的进步。通过结合不同领域的这些技术突破，我们希望在没有晶体的晶体学上取得成功。该项目具有转移晶体学范式的潜力。尽管此阶段的辐射源是脉冲相干电子束，但基本的操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在梁线上建立，例如斯坦福线性加速器中心或国家同步灯源II。通过将电喷雾电离源的占空比与辐射源的占空比匹配，可以将样品消耗减少到Femtomoles，即使是最困难的蛋白质也可以实现的目标。随着大分子，蛋白质复合物和纳米材料的喷涂技术进一步发展，晶体生长的困难但不可预测的过程将不再是强制性的。通过大量节省的人力，时间和金钱，可以及时且具有成本效益的方式从结构信息中测试疾病机制和激进治疗策略的大胆假设。仅仅是因为人们不能证明需要大量的时间和精力来种植足够大的单晶以获得结构证据所需的大量时间和精力，因此不再需要驳回一个想法。因此，减少或消除晶体学的尺寸限制可以在生物科学家的心态中带来根本的转变。 公共卫生相关性：如果成功的话，该项目有可能大大加快包括癌症，艾滋病和糖尿病在内的疾病的机械和治疗研究率。药物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，该项目将消除晶体学对单晶的依赖。通过大量节省的人力，时间和金钱，可以及时且具有成本效益的方式从结构信息中测试疾病机制和激进治疗策略的大胆假设。