Atomic resolution protein structures from electron diffraction of oriented ions

通过定向离子的电子衍射获得原子分辨率的蛋白质结构

• 批准号: 9066716
• 负责人: Wei Kong
• 金额: $ 26.23万
• 依托单位: OREGON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9066716
• 关键词: Address; Anisotropy; Binding; Biological; Biophysics; Charge; Chills; Collection; Complex; Country; Crystallization; Crystallography; Data; Data Quality; Detection; Development; Discipline; Electron Beam; Electron Diffraction Microscopy; Electrons; Electrospray Ionization; Freezing; Future; Gases; Generations; Genome; Goals; Grant; Health; Heating; Helium; Hour; Human Genome; Image; Individual; Integral Membrane Protein; Investments; Ions; Knowledge; Laboratories; Lasers; Ligand Binding; Light; Maps; Mass Spectrum Analysis; Methods; Molecular; Molecular Conformation; Molecular Structure; Mutation; Optics; Organic solvent product; Pharmaceutical Preparations; Phase; Physiologic pulse; Preclinical Drug Evaluation; Process; Property; Proteins; Radiation; Resolution; Rest; Sampling; Solvents; Structure; System; Technology; Temperature; base; cryogenics; design; disease-causing mutation; electric field; electron density; electron diffraction; imaging system; innovation; insight; instrument; macromolecule; millisecond; next generation; novel strategies; protein complex; protein folding; protein structure; single molecule; structural biology

The human genome has been sequenced for a decade, but solving how proteins fold and assemble into complexes remains a challenge. More than half of all proteins -- including 95% of integral membrane proteins -- do not crystallize and thus their structures cannot be determined by crystallography. Our project addresses this problem by creating an instrument that can determine atomic-resolution structures of individual biological macromolecules without requiring crystallization. We propose to merge four distinct technologies that should allow structures of macromolecules up to a megaDalton to be resolved at high resolution (better than 2 ¿) in a few hours. The key steps are a) to electrospray and purify macromolecules by mass spectrometry, b) to quickly chill these macromolecules to near absolute zero temperature with superfluidic helium droplets, c) to controllably orient several thousand chilled macromolecules to within ~1¿ for 50 ¿s using intense elliptically polarized IR laser light while confining them in a small "diffraction" zone, and d) to collect continuous diffraction images from these oriented macromolecules using a pulsed electron beam. Steps c) and d) will be repeated for each orientation to span the reciprocal space at 1¿ intervals by rotating the polarization of the laser. The continuous diffraction images provide sufficient information to directly calculate phases by well-established oversampling methods thereby directly yielding electron density maps. In this grant period, our goal is to demonstrate the proof-of-concept by recording anisotropic electron diffraction images from laser aligned protein ions embedded in superfluid helium droplets. Further development will address the resolution and quality of data issues with major improvements in experimental hardware. This idea is based on recent breakthroughs in several disciplines. A large body of evidence has established that protein complexes can retain their conformation, remain associated in large multimeric complexes and keep ligands bound in vacuo after electrospray ionization. Capitalizing on recent advances in laser-induced alignment at superfluid helium temperatures (0.37 Kelvin), our proposed instrument will instantaneously freeze macromolecules, allowing them to be oriented within 1¿ in all three Euler angles by a 200,000 V/cm electric field generated by the IR laser. Ultimately, this approach will allow structures to be determined at high resolution in a few hours from a few nanomoles of partially purified complexes of proteins that are otherwise inaccessible by current methods. If successful, this instrument will reshape the landscape of structural biology, transform structure-based drug screening, allow rapid determination of the effects of mutations on structure, and open new realms of biophysics to understand the effects of solvent on structure.

人类基因组已经测序了十年，但是解决了蛋白质如何折叠并组装成 复合物仍然是一个挑战。所有蛋白质的一半以上 - 包括95％的整体膜 蛋白质 - 不要结晶，因此无法通过晶体学确定其结构。我们的 项目通过创建可以确定原子分辨率结构的工具来解决此问题 单个生物学大分子无需结晶。我们建议合并四个 不同的技术应允许大分子的结构达到梅加达尔顿 几个小时内高分辨率（大于2€）。关键步骤是a）电喷雾和净化 大分子通过质谱法，b）将这些大分子迅速冷却至绝对零。 用超氟氦液滴的温度，c）控制地定向数千条冷藏 大分子在50 s内使用强烈的椭圆极化的红外激光，而限制 它们在一个小的“衍射”区域，d）从这些方向收集连续的衍射图像 大分子使用脉冲电子束。步骤c）和d）将重复每个方向 通过旋转激光的极化，以1范围的间隔跨越相互空间。连续 衍射图像提供了足够的信息，可以直接计算出良好的 过采样方法，因此直接得出电子密度图。在这个赠款期间，我们的目标是 通过记录各向异性电子衍射图像来证明概念的证明 嵌入超流体氦液滴中的蛋白质离子。进一步发展将解决该决议 数据问题的质量以及实验硬件的重大改进。这个想法是基于 最近在几个学科中的突破。大量证据已经确定了蛋白质 复合物可以保留其构象，保持在大型的多个综合体中，并保持 电喷雾电离后与真空化结合的配体。利用激光诱导的最新进展 在超流体氦气温度（0.37 kelvin）处对齐，我们的拟议仪器将立即 冻结大分子，使它们在所有三个Euler角度以200,000 IR激光器产生的V/cm电场。最终，这种方法将使结构成为 从几个小时内从几个纳莫尔的几个纳莫尔的部分纯化的复合物中确定 蛋白质是通过当前方法无法访问的。如果成功，该乐器将重塑 结构生物学的景观，基于结构的药物筛查，可以快速确定 突变对结构的影响，并开放新的生物物理学领域，以了解 结构解决方案。