Technology development for biological imaging with x-ray free electron lasers

测定大型蛋白质复合体的结构并对其动力学行为进行成像 与其他在室温下具有接近原子分辨率的生物纳米颗粒一样， 有可能极大地影响结构生物学和我们对生物分子功能和 互动。结构生物学的一个主要瓶颈是许多蛋白质 关键的细胞功能是膜蛋白，它被证明是结构上难以处理的 由传统的x射线结晶学测定，其中x射线辐射损伤通过以下方式减轻 将辐射剂量分散到晶体中的许多分子上。因此，大多数膜 到目前为止，蛋白质结构仍然未知。同样，确定高分辨率的x射线结构 单一的、非周期的生物纳米颗粒的X射线衍射成像受到以下因素的阻碍 辐射损伤。虽然冷冻电子显微镜(Cryo-EM)已经成功地获得了 来自大的生物分子和纳米颗粒的高分辨率结构信息，它需要 冷冻样品作为减轻电子诱导辐射损伤的一种方式和低温 温度使得人们不可能看到快速的构象变化。 产生超短和超亮X射线脉冲的X射线自由电子激光(XFELs)允许 要打破分辨率和吸收剂量之间的这种联系，需要利用“先衍射后吸收”的方法。 破坏“的原则和承诺以前所未有的时空分辨率成像。自.以来 SLAC国家加速器直线加速器相干光源的调试 实验室仅仅五年前，两种蛋白质的结构测定都是在常温下进行的 串飞秒纳米晶体(SFX)的原子分辨率和中等分辨率 利用单纳米粒子衍射成像(SPI)对其结构进行了研究。 然而，仍有一些挑战和限制需要克服，以便更充分地 利用这些新光源进行结构生物学。 这项拟议工作的总体目标是解决当前的几项技术和 用XFELS对生物样品进行相干x射线衍射成像的方法学挑战， 特别是在膜蛋白和膜蛋白的样品制备和引入领域。 生物纳米颗粒。我们的工作旨在大幅减少样品消耗，并开放 这种成像方法适用于范围更广的研究小组和样本，包括 膜蛋白和其他生物纳米物体的多样性 难以结晶的。拟议的工作将为未来的时间解决奠定基础 要求有效利用现有样品的XFELs结构研究。 如果成功，这项工作将极大地帮助我们的实验能力来学习和理解 蛋白质复合体和生物纳米颗粒在广泛领域的功能，包括 人类健康和生物安全。

Determining the structure and imaging the dynamical behavior 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 many of the proteins performing critical cellular functions are membrane proteins that 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. Similarly, determining high-resolution x-ray structures of single, non-periodic biological nanoparticles by x-ray diffraction imaging has been hampered by radiation damage. 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 as a way to mitigate electron-induced radiation damage and the cryogenic temperatures make it impossible to visualize fast conformational changes. X-ray free electron lasers (XFELs), which produce ultra-short and ultra-bright x-ray pulses, allow to break this nexus between resolution and absorbed dose by utilizing the “diffraction-before- destruction” principle and promise imaging at unprecedented spatio-temporal resolution. Since the commissioning of the Linac Coherent Light Source (LCLS) at SLAC National Accelerator Laboratory only five years ago, both protein structure determination at room temperature to near- atomic resolution by serial-femtosecond nanocrystallography (SFX) and modest resolution structural studies by single nanoparticle diffraction imaging (SPI) have been demonstrated. However, several challenges and limitations remain that need to be overcome to allow more fully utilizing these new light sources for structural biology. The overall objective of this proposed work is to address several of the current technological and methodological challenges in coherent x-ray diffraction imaging of biological samples with XFELs, in particular, in the areas of sample preparation and introduction for membrane proteins and biological nanoparticles. Our work aims to drastically reduce sample consumption and to open up this imaging method to a much broader range of research groups and to samples including a diversity of membrane proteins and other biological nano-objects that are not abundant and/or difficult to crystallize. The proposed work will lay the groundwork for future time-resolved structural studies at XFELs that require efficient use of available sample. 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.