Combining native protein mass spectrometry with serial electron diffraction to solve atomic structures of mass selected macromolecules

将天然蛋白质质谱与串行电子衍射相结合来解析质量选择的大分子的原子结构

• 批准号: 10637752
• 负责人: Wei Kong
• 金额: $ 82.46万
• 依托单位: OREGON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-11 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10637752
• 关键词: Address; Biological; Cations; Cells; Charge; Collection; Computer software; Data; Data Analyses; Disease; Drug Screening; Electron Beam; Electrons; Electrospray Ionization; Electrostatics; Elements; Explosion; Foundations; Freezing; Goals; Guns; Helium; Heterogeneity; Individual; Ions; Label; Lasers; Ligands; Mass Spectrum Analysis; Membrane Proteins; Methods; Microfluidics; Molecular; Molecular Conformation; Mutation; Noise; Pattern; Pharmaceutical Preparations; Phase; Physiologic pulse; Process; Property; Proteins; Pyrenes; Resolution; Retrieval; Sampling; Shapes; Signal Transduction; Source; Specialist; Structure; Technology; Temperature; Therapeutic; Time; computerized data processing; cryogenics; density; design; detector; digital; dimer; dipole moment; drug discovery; electric field; electron diffraction; high risk; innovation; instrument; ion source; macromolecule; mass spectrometer; member; nano-electrospray; nanocrystal; nanoionics; nanomaterials; particle; protein complex; protein protein interaction; public health relevance; recruit; risk mitigation; small molecule; structural biology

We plan to add an electron diffraction component to a native protein mass spectrometer to create a new instrument that can derive atomic structures of macromolecules such as proteins. The key innovation is the use of superfluid helium droplets for sample cooling thereby effective field induced orientation and alignment. Mass and conformation selected proteins from a native electrospray ionization mass spectrometer are embedded in superfluid helium droplets, and in a pulsed electric field and elliptically polarized laser field, due to the permanent and induced dipoles of the protein, all three Euler angles of the protein can be precisely defined. The large polarizability volume of macromolecules (not the permanent dipole moment) and the low rotational temperature of the embedded macromolecules are the two elements of the “molecular goniometer”: changing the polarization properties of the laser field changes the orientation of the macromolecule. Electron diffraction patterns from macromolecule-doped droplets, one molecule per droplet, all oriented in the same direction, are accumulated with each successive pulse, until a satisfactory signal-to-noise ratio is achieved. Ultimately from the diffraction patterns of all orientations of the chosen macromolecule, the electrostatic potential is derived using the oversampling method for iterative phase retrieval and structure determination. In the past few years, we have accumulated preliminary data on electron diffraction of small molecules and cationic molecular clusters embedded in superfluid helium droplets, and on doping macromolecular ions into superfluid helium droplets. The next phase of the project is to construct a complete instrument to demonstrate the principle of the concept. We now can solve structures of nanocrystals embedded in superfluid helium droplets, both neutral and charged, without sample alignment. Therefore the background issue of the enclosing helium and the particle density issue of charged species are no longer major concerns. Our demonstrated resolution from pyrene dimer cations is 0.5 Å. Moreover, we have succeeded in doping macromolecular ions into superfluid helium droplets using a standard electrospray ionization source. Our accomplishments so far have laid the foundation for the next phase of progress, and we are now ready to demonstrate the principle of the concept. With the acquisition of a new electron gun, a upgrade to a native protein ion source, and a direct electron detector, we have a detailed plan to align all three pulsed beams, the laser beam, the ion doped droplet beam, and the electron beam, to obtain diffraction patterns of field aligned macromolecules. Our ultimate goal is to resolve atomic structures of mass and conformation selected macromolecules with 1 Å resolution from mixtures of protein solutions, microfluidic reactors, or labeled cells for proteins and protein complexes. The final instrument will reshape the landscape of structural biology, transform structure-based drug screening, and rapidly determine effects of mutations and deletions on structure. It will also offer structural assessment of components in polydisperse mixtures of nanomaterials important for biomedical applications. To mitigate the risks, we have recruited a specialist in mass spectrometry, Dr. David Russell, to be our consultant, and a specialist in data processing, Dr. Peter Schwander, to be a member of our team.

我们计划在天然蛋白质质谱仪中添加电子衍射组件，以创建一种新仪器 可以推导出蛋白质等大分子的原子结构。关键的创新是使用超流氦 用于样品冷却的液滴，从而有效的场诱导定向和对准。选择的质量和构象 来自天然电喷雾电离质谱仪的蛋白质被嵌入超流氦液滴中，并在 脉冲电场和椭圆偏振激光场，由于蛋白质的永久偶极子和感应偶极子，所有三个 可以精确定义蛋白质的欧拉角。大分子的极化率体积大（不是 永久偶极矩）和嵌入大分子的低旋转温度是两个要素 “分子测角仪”：改变激光场的偏振特性会改变激光场的方向 高分子。来自高分子掺杂液滴的电子衍射图案，每个液滴一个分子，全部定向 在同一方向上，每个连续脉冲都会累积，直到达到满意的信噪比。 最终，根据所选大分子所有方向的衍射图案，静电势为 使用过采样方法进行迭代相位检索和结构确定。 在过去的几年里，我们积累了小分子和阳离子电子衍射的初步数据。 嵌入超流氦液滴中的分子簇以及将大分子离子掺杂到超流氦中 飞沫。该项目的下一阶段是构建一个完整的仪器来演示该概念的原理。 我们现在可以解析嵌入超流氦液滴（中性和带电）中的纳米晶体的结构，而无需 样本对齐。因此，封闭氦气的背景问题和带电粒子密度问题 物种不再是主要问题。我们证明芘二聚体阳离子的分辨率为 0.5 Å。此外，我们 使用标准电喷雾电离成功地将大分子离子掺杂到超流氦液滴中 来源。目前我们所取得的成绩已经为下一阶段的进展奠定了基础，我们现在已经准备好 展示概念的原理。随着新电子枪的获得，升级为天然蛋白质离子 源和直接电子探测器，我们有一个详细的计划来对齐所有三个脉冲束，激光束，离子束 掺杂液滴束和电子束，以获得场排列大分子的衍射图案。 我们的最终目标是以 1 Å 分辨率解析质量和构象选定的大分子的原子结构 来自蛋白质溶液的混合物、微流体反应器或蛋白质和蛋白质复合物的标记细胞。决赛 仪器将重塑结构生物学的格局，改变基于结构的药物筛选，并迅速 确定突变和缺失对结构的影响。它还将提供组件的结构评估 纳米材料的多分散混合物对于生物医学应用很重要。为了降低风险，我们招募了一名 质谱专家 David Russell 博士担任我们的顾问，数据处理专家 Peter 博士 施万德，成为我们团队的一员。