Technology Development for Biological Imaging with XFELs

项目摘要/摘要 测定大型蛋白质复合体的结构和构象动力学以及其他 在室温下具有接近原子分辨率的生物纳米颗粒有可能极大地影响 结构生物学和我们对生物分子功能和相互作用的知识。一个主要的瓶颈是 结构生物学是，虽然许多关键的细胞功能是由膜蛋白执行的，但它们 已被证明难以用传统的x射线结晶学进行结构测定，其中x射线辐射 通过将辐射剂量分散到晶体中的许多分子上，可以减轻损害。因此，大多数 到目前为止，膜蛋白结构仍然未知。而冷冻电子显微镜(Cryo-EM)已经 成功地从大的生物分子和纳米粒子中获得高分辨率的结构信息，它 需要冷冻样品以减轻电子诱导的辐射损害和低温测量 使得我们不可能看到快速的构象变化。 X射线自由电子激光(XFELs)产生超短和超亮的X射线脉冲，使我们能够 利用“先衍射后销毁”来打破分辨率和辐射损伤之间的联系 以前所未有的时空分辨率进行成像的原理和前景。在过去的十年里， 世界上第一个XFEL，直线加速器相干光源(LCLS)在SLAC国家加速器上开放 实验室，在室温下进行蛋白质结构测定，达到近原子分辨率，通过串联- 飞秒纳米结晶术(SFX)已经被证明。然而，一些挑战和限制 仍然需要解决，以充分利用这些新光源和即将到来的 用于结构生物学的下一代XFELs。 这项提案的总体目标是通过解决当前的几个 利用XFELs对生物样品进行X射线衍射成像的技术和方法挑战 特别是在膜蛋白的样品制备领域，通常膜蛋白的丰度较低 和/或难以结晶，能够实现高数据采集率的样品引入技术，以及新颖的 膜蛋白的时间分辨结构测定方法。这项工作也将极大地 减少样品消耗，将增加膜蛋白等生物纳米的多样性 可以使用XFEL研究的对象。拟议的工作还旨在开发新的时间能力- XFELs解析结构研究包括跨膜电位触发的膜蛋白 动力学，使研究更广泛的生物分子和细胞反应以及相关的 在很大的时间范围内，结构变化从微秒到毫秒不等。如果成功，这项工作 将极大地帮助我们的实验能力来研究和了解蛋白质复合体的功能 生物纳米颗粒在包括人类健康和生物安全在内的广泛领域。

Project Summary / Abstract Determining the structure and conformational dynamics of large protein complexes as well as other biological nanoparticles at room temperature with near atomic resolution has the potential to greatly impact structural biology and our knowledge of biomolecular function and interactions. A major bottleneck in structural biology is that while many critical cellular functions are performed by membrane proteins, they have proven intractable to structure determination by traditional x-ray crystallography, in which x-ray radiation damage is mitigated by spreading the radiation dose over many molecules in a crystal. Consequently, most membrane protein structures remain unknown to date. While cryo-electron microscopy (cryo-EM) has been successful in obtaining high-resolution structural information from large biomolecules and nanoparticles, it requires freezing of the sample to mitigate electron-induced radiation damage and cryogenic measurement makes it impossible to visualize fast conformational changes. X-ray free electron lasers (XFELs), which produce ultra-short and ultra-bright x-ray pulses, allow us to break this nexus between resolution and radiation damage by utilizing the “diffraction-before-destruction” principle and promise imaging at unprecedented spatio-temporal resolution. Over the last decade since the opening of the world's first XFEL, the Linac Coherent Light Source (LCLS) at SLAC National Accelerator Laboratory, protein structure determination at room temperature to near-atomic resolution by serial- femtosecond nanocrystallography (SFX) has been demonstrated. However, several challenges and limitations remain that need to be addressed to fully utilize the capabilities of these new light sources and the upcoming next generation XFELs for structural biology. The overall objective of this proposal is to enable new science by addressing several of the current technological and methodological challenges in x-ray diffractive imaging of biological samples with XFELs, in particular in the areas of sample preparation for membrane proteins that, generally, suffer from low abundance and/or are hard to crystallize, sample introduction technologies enabling high data acquisition rates, and novel approaches to time-resolved structure determination of membrane proteins. This work will also drastically reduce sample consumption and will increase the diversity of membrane protein and other biological nano- objects that can be studied with XFELs. The proposed work also aims to develop new capabilities for time- resolved structural studies at XFELs to include cross-membrane potential triggered membrane protein dynamics, enabling investigation of a broader range of biomolecular and cellular reactions and the associated structural change over a large range of times scales from microseconds to milliseconds. If successful, this work would greatly aid our experimental capabilities to study and understand function of protein complexes and biological nanoparticles in a wide range of fields including human health and biosecurity.