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

确定大蛋白质复合物的结构并对其动力学行为进行成像 与室温下具有接近原子分辨率的其他生物纳米颗粒一样， 可能会极大地影响结构生物学和我们对生物分子功能的认识， 交互.结构生物学中的一个主要瓶颈是，许多蛋白质的功能 关键的细胞功能是膜蛋白，已被证明难以结构化 通过传统的X射线晶体学进行测定，其中通过 将辐射剂量分散到晶体中的许多分子上。因此，大多数膜 蛋白质结构至今仍是未知的。同样，确定高分辨率的x射线结构 单一的，非周期性的生物纳米粒子的x射线衍射成像已经受到阻碍， 辐射损伤虽然冷冻电子显微镜（cryo-EM）已经成功地获得了 从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， 温度使得不可能观察到快速的构象变化。 X射线自由电子激光器（XFEL）产生超短和超亮的X射线脉冲， 为了打破分辨率和吸收剂量之间的这种联系， 破坏”的原则和承诺成像在前所未有的时空分辨率。以来 直线加速器相干光源（LCLS）在SLAC国家加速器的调试 实验室仅在五年前，就在室温下测定了两种蛋白质的结构， 通过连续飞秒纳米照相术（SFX）的原子分辨率和中等分辨率 已经证明了通过单纳米颗粒衍射成像（SPI）的结构研究。 然而，仍有一些挑战和限制需要克服， 利用这些新光源进行结构生物学研究。 这项拟议工作的总体目标是解决目前的几个技术和 用XFEL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， 这种成像方法适用于更广泛的研究小组和样本，包括 - 膜蛋白和其他生物纳米物体的多样性，这些生物纳米物体并不丰富和/或 很难结晶。拟议的工作将为今后的时间分辨 需要有效利用现有样品的XFEL结构研究。 如果成功，这项工作将大大有助于我们的实验能力，研究和理解 蛋白质复合物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.