Auer M
Skirball Institute of Biomolecular Medicine, New York University Medical Center, NY 10016, USA.
J Mol Med (Berl). 2000;78(4):191-202. doi: 10.1007/s001090000101.
Electron cryo-microscopy has established itself as a valuable method for the structure determination of protein molecules, protein complexes, and cell organelles. This contribution presents an introduction to the various aspects of three-dimensional electron cryomicroscopy. This includes the need for sample preservation in the microscope vacuum, strategies for minimizing radiation damage, methods of improving the poor signal-to-noise ratio in electron micrographs of unstained specimens, and the various methods of three-dimensional image reconstruction from projections. The various specimen types (e.g., flat and tubular two-dimensional crystals, protein filaments, individual protein molecules, and large complexes) require different means of three-dimensional reconstruction, and we review the five major reconstruction techniques (electron crystallography, helical reconstruction, icosahedral reconstruction, single-particle reconstruction, and electron tomography), with an emphasis on electron crystallography. Several medically relevant three-dimensional protein structures are chosen to illustrate the potential of electron cryo-microscopy and image reconstruction techniques. Among the structural methods, electron cryo-microscopy is the only tool for studying objects that range in size from small proteins over macromolecular complexes to cell organelles or even cells.
电子冷冻显微镜已成为确定蛋白质分子、蛋白质复合物和细胞器结构的一种重要方法。本文介绍了三维电子冷冻显微镜的各个方面。这包括在显微镜真空中保存样品的必要性、使辐射损伤最小化的策略、改善未染色标本电子显微照片中信噪比低的方法，以及从投影进行三维图像重建的各种方法。不同类型的标本（如扁平及管状二维晶体、蛋白质细丝、单个蛋白质分子和大型复合物）需要不同的三维重建方法，我们综述了五种主要的重建技术（电子晶体学、螺旋重建、二十面体重建、单颗粒重建和电子断层扫描），重点是电子晶体学。选择了几种与医学相关的三维蛋白质结构来说明电子冷冻显微镜和图像重建技术的潜力。在各种结构方法中，电子冷冻显微镜是研究从小蛋白质到大分子复合物，再到细胞器甚至细胞等不同大小物体的唯一工具。
