Abstract：
Microcrystal electron diffraction (MicroED) enables atomic resolution structures to be determined from vanishingly small crystals. Soluble proteins typically grow crystals that are tens to hundreds of microns in size for X-ray crystallography. But membrane protein crystals often grow crystals that are too small for X-ray diffraction and yet too large for MicroED. These crystals are often formed in thick, viscous media that challenge traditional cryoEM grid preparation. Here, we describe two approaches for preparing membrane protein crystals for MicroED data collection: application of a crystal slurry directly to EM grids, and focused ion beam milling in a Scanning Electron Microscope (FIB-SEM). We summarize the case of preparing an ion channel, NaK, and the workflow of focused ion-beam milling. By milling away the excess media and crystalline material, crystals of any size may be prepared for MicroED. Finally, an energy filter may be used to help minimize inelastic scattering leading to lower noise on recorded images.
摘要：
微晶电子衍射（MicroED）可以从消失的小晶体中确定原子分辨率结构。可溶性蛋白质通常生长出用于X射线晶体学的数十至数百微米大小的晶体。但是膜蛋白晶体通常生长的晶体对于X射线衍射来说太小，而对于MicroED来说太大。这些晶体通常形成厚厚的，挑战传统的cryoEM网格制备的粘性介质。这里,我们描述了两种制备用于MicroED数据收集的膜蛋白晶体的方法：将晶体浆液直接应用于EM网格，和聚焦离子束铣削在扫描电子显微镜（FIB-SEM）。我们总结了准备离子通道的情况，NaK,以及聚焦离子束铣削的工作流程。通过碾磨掉多余的介质和晶体材料，可以为MicroED制备任何尺寸的晶体。最后,能量滤波器可用于帮助最小化非弹性散射，从而降低记录图像上的噪声。
