Atomic resolution protein structures from electron diffraction of oriented ions

人类基因组测序已经进行了十年，但解决蛋白质如何折叠和组装成 复合体仍然是一个挑战。超过一半的蛋白质-包括95%的膜完整蛋白质 蛋白质不结晶，因此它们的结构不能通过晶体学确定。我们 一个项目通过创建一个可以确定原子分辨率结构的仪器来解决这个问题 而不需要结晶。我们建议合并四个 不同的技术，应该允许大分子的结构高达百万道尔顿，以解决在 高分辨率（优于2 º）在几个小时内。关键步骤是a）电喷雾和纯化 B）将这些大分子快速冷却至接近绝对零度 c）以可控地定向数千个冷却的 使用强烈的椭圆偏振IR激光将大分子限制在~1 <$$>内50 <$s，同时限制 d）从这些定向的衍射图像中收集连续的衍射图像， 使用脉冲电子束对大分子进行化学修饰。将针对每个方向重复步骤c）和d）， 通过旋转激光的偏振，以1？的间隔跨越倒易空间。连续 衍射图像提供了足够的信息来直接计算相位 过采样方法，从而直接产生电子密度图。在这段时间里，我们的目标是 通过记录来自激光对准的各向异性电子衍射图像来证明概念验证 蛋白质离子嵌入超流氦滴中。进一步的发展将解决该决议 和数据质量的问题与实验硬件的重大改进。我们的建议是基于 最近几个学科的突破。大量的证据表明， 复合物可以保持它们的构象，在大的多聚体复合物中保持缔合， 电喷雾电离后真空结合配体。利用激光诱导 在超流氦温度（0.37开尔文）下对准，我们提出的仪器将立即 冻结大分子，使它们在所有三个欧拉角中的1 ½内定向， 由IR激光器产生的V/cm电场。最终，这种方法将允许结构 在几个小时内从几纳摩尔的部分纯化的复合物中以高分辨率测定， 这些蛋白质是目前的方法无法获得的。如果成功，这一工具将重塑 结构生物学前景，改变了基于结构的药物筛选，允许快速确定 突变对结构的影响，并开辟了生物物理学的新领域，以了解 结构上的溶剂。

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.