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.

我们计划将电子衍射成分添加到天然蛋白质质谱仪中，以创建一种新仪器 可以得出大分子（例如蛋白质）的原子结构。关键的创新是使用超流体氦气 用于样品冷却的液滴，从而有效场引起方向和比对。选择的质量和构象 来自天然电喷雾电离质谱仪的蛋白质嵌入在超流体氦液滴中，并在A中 由于蛋白质的永久性和诱导的偶极子，脉冲电场和椭圆极化激光场 可以精确定义蛋白质的欧拉角。大分子的极化量较大（不是 永久偶极矩）和嵌入式大分子的低旋转温度是 “分子角仪”：改变激光场的极化特性改变了 高分子。来自大分子掺杂的液滴的电子衍射图，每滴一个分子，全部取向 在同一方向上，每个成功的脉冲都积累，直到达到令人满意的信噪比为止。 最终，从所选大分子的所有方向的衍射模式中，静电电势是 使用过度采样方法得出迭代相的检索和结构确定。 在过去的几年中，我们积累了有关小分子和阳离子电子衍射的初步数据 分子簇嵌入了超流体氦液滴中，并将大分子离子掺入超氟氦 飞沫。该项目的下一个阶段是构建一种完整的工具来展示该概念的原理。 现在，我们可以解决嵌入中性和充电的超富集氦液滴中的纳米晶体的结构，没有 样品比对。因此，封闭氦气的背景问题和带电的粒子密度问题 物种不再是主要问题。我们从pyrene二聚体阳离子中证明的分辨率为0.5Å。而且，我们 使用标准电喷雾电离成功地将大分子离子掺入超流体氦液滴中 来源。到目前为止，我们的成就为下一阶段的进步奠定了基础，我们现在准备好 演示该概念的原则。随着新电子枪的获取，对天然蛋白离子的升级 来源和直接电子检测器，我们制定了一个详细的计划，将所有三个脉冲梁，激光束，离子对齐 掺杂的液滴束和电子束，以获得场对齐大分子的衍射图。 我们的最终目标是解决以1Å分辨率的质量和成分的原子结构 来自蛋白质溶液的混合物，微流体反应器或蛋白质复合物的标记细胞。决赛 仪器将重塑结构生物学的景观，转化基于结构的药物筛查并迅速 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。为了减轻风险，我们招募了 质谱专家David Russell博士是我们的顾问，并且是数据处理专家Peter博士 Schwander，成为我们团队的成员。