Atomic resolution protein structures from electron diffraction of oriented ions

人类基因组已经被测序了十年，但解决了蛋白质如何折叠和组装成 建筑群仍然是一个挑战。超过一半的蛋白质--包括95%的完整细胞膜 蛋白质--不结晶，因此其结构不能通过结晶学来确定。我们的 Project通过创建一种可以确定原子分辨率结构的仪器来解决这个问题 不需要结晶的单个生物大分子。我们建议将四家公司合并 独特的技术，应该允许高达一兆吨的大分子结构以 几小时内即可获得高分辨率(优于2？)。关键步骤是a)电喷雾和净化 通过质谱仪检测大分子，b)将这些大分子快速冷却到接近绝对零度 超流氦液滴的温度，c)可控定向数千次冷却 S利用强椭圆偏振红外激光在禁闭时将大分子限制在~1°内 D)收集来自这些定向的连续的衍射图像 使用脉冲电子束的大分子。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。不间断的 衍射图提供了足够的信息来直接计算相 过采样方法，从而直接产生电子密度图。在这一批款期内，我们的目标是 通过记录激光对准的各向异性电子衍射图像来演示概念验证 嵌入在超流氦液滴中的蛋白质离子。进一步的发展将解决该决议 以及实验硬件的重大改进带来的数据质量问题。这个想法是基于 最近在几个学科上取得了突破。大量的证据已经证实了这种蛋白质 络合物可以保持其构象，保持在大的多聚体络合物中，并保持 电喷雾电离后，配体结合在真空中。利用激光诱导技术的最新进展 在超流氦温度(0.37开尔文)下对准，我们提出的仪器将瞬间 冻结大分子，允许它们在所有三个欧拉角的1？内定向200,000 红外激光产生的V/cm电场。最终，这种方法将允许结构 从几个纳米分子的部分纯化的络合物在几个小时内以高分辨率测定 目前的方法无法获得的蛋白质。如果成功，这一工具将重塑 结构生物学的景观，转变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.