Tools for Enabling Challenging Structure Determination by Cryo-EM

摘要 最近的技术革新大大提高了低温电子 显微镜（cryo-EM），导致结构数量的增殖， 分辨率随着许多“低挂水果”结构的确定，研究已经转移到 越来越复杂的标本，推动当前处理软件的能力， 需要新颖的方法。冷冻EM的一类挑战性分子是管状分子， 可以有模糊的对称性。特别是，形成小管的膜重塑蛋白， 通常仅仅是局部有序的，这使得它们难以进行结构确定。面临的另一个挑战 cryo-EM结构确定是空气/水界面的问题。通常在印迹样品时 对于冷冻EM制备，样品可以与空气/水界面相互作用， 变性、聚集或优选取向。我们开发了一种新技术， 调用平均减去管状区域（RASTR）的重建，其具有解决 这两个问题。RASTR将管状冷冻EM样品分解成单个表面， 使得能够在不知道或应用螺旋对称性的情况下确定结构， 仅局部有序而无长程有序的管状样品。在此，我们提出两个目标， 使RASTR更健壮、更通用、更高分辨率。这些目标将由 具有挑战性的管状样品已被证明是典型的低温EM结构难以处理的 测定方法最后，第三个目标是将RASTR方法扩展到单粒子束缚 以保护它们不受空气/水界面的影响并防止优选的 方向。这些拟议的目标共同赋予了RASTR成为一个通用平台的潜力， 能够对具有挑战性的样品进行结构测定。

Abstract Recent technological innovations have significantly increased the efficiency of cryogenic electron microscopy (cryo-EM), causing a proliferation in the number of structures resolved with ever-increasing resolution. As many of the "low hanging fruit" structures have been determined, studies have shifted toward increasingly complex specimens, pushing the capabilities of current processing software and requiring novel methodologies. One class of challenging molecule for cryo-EM is tubular molecules that can have ambiguous symmetries. In particular, membrane remodeling proteins that form tubules are often only locally ordered making them intractable for structure determination. Another challenge for cryo-EM structure determination is the problem of the air/water interface. Often when blotting a sample for cryo-EM preparation, the sample can interact with the air/water interface causing protein denaturation, aggregation, or preferred orientations. We have developed a new technique that we are calling reconstruction of average subtracted tubular regions (RASTR) that has the potential to solve both of these problems. RASTR breaks down tubular cryo-EM samples into individual surfaces which enables structure determination without knowing or applying helical symmetry and classification of tubular samples that are only locally ordered with no long-range order. Here we propose two aims to make RASTR more robust, generalizable, and higher-resolution. These aims will be driven by challenging tubular samples that have proven to be intractable to typical cryo-EM structure determination methods. Finally, a third aim is to extend the RASTR approach to single particles bound to tubular substrates in order to protect them from the air/water interface and prevent preferred orientations. Together the proposed aims give RASTR the potential to become a general platform to enable structure determination of challenging samples.