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

我们计划在天然蛋白质质谱计上增加一个电子衍射元件，以创造一种新的仪器， 可以推导出蛋白质等大分子的原子结构。关键的创新是超流氦的使用。 用于样品冷却的液滴，从而有效地场诱导取向和取向。选定的质量和构象 来自本地电喷雾电离质谱仪的蛋白质被嵌入到超流氦液滴中，并在 脉冲电场和椭圆偏振激光场，由于蛋白质的永久和诱导偶极，这三个都是 可以精确地定义蛋白质的欧拉角。大分子的大的极化体积(不是 永久偶极矩)和嵌入的大分子的低旋转温度是 分子测角仪：改变激光场的偏振属性改变方向 大分子。大分子掺杂液滴的电子衍射图，每个液滴一个分子，都是定向的 在相同方向上，随着每个连续脉冲累积，直到获得令人满意的信噪比。 最终，从所选大分子所有取向的衍射图来看，静电势为 采用过采样法进行迭代相位恢复和结构确定。 在过去的几年里，我们积累了小分子和阳离子的电子衍射的初步数据。 嵌入在超流氦液滴中的分子团簇以及大分子离子在超流氦中的掺杂 水滴。该项目的下一阶段是构建一套完整的工具来演示这一概念的原理。 我们现在可以解决嵌入在超流氦液滴中的纳米晶体的结构，无论是中性的还是带电的，而不需要 样本对齐。因此，封闭氦的背景问题和带电粒子的密度问题 物种不再是主要关注的问题。我们展示的来自二聚芘阳离子的分辨率为0.5？此外，我们 已经成功地使用标准的电喷雾电离将大分子离子掺杂到超流氦液滴中 消息来源。我们迄今取得的成就为下一阶段的进展奠定了基础，我们现在准备 说明该概念的原理。随着获得新的电子枪，升级到天然蛋白质离子 光源和直接电子探测器，我们有一个详细的计划来对准所有三个脉冲光束，激光光束，离子 掺杂液滴束和电子束，以获得场定向大分子的衍射图。 我们的最终目标是以1？分辨率解析选定的大分子的质量和构象的原子结构 来自蛋白质溶液、微流控反应器的混合物，或蛋白质和蛋白质复合体的标记细胞。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。它还将提供对以下组件的结构评估 对生物医学应用非常重要的纳米材料的多分散混合物。为了降低风险，我们聘请了一名 质谱学专家David Russell博士将成为我们的顾问，数据处理专家Peter博士将成为我们的顾问 施万德，成为我们团队的一员。

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.