A giant technological leap in the field of cryo-electron microscopy (cryo-EM) has assured the achievement of near-atomic resolution structures of biological macromolecules. As a recognition of this accomplishment, the Nobel Prize in Chemistry was awarded in 2017 to Jacques Dubochet, Joachim Frank, and Richard Henderson, the pioneers in the field of cryo-EM. Currently, the technique has become the method of choice for structural analysis of heterogeneous and intrinsically dynamic biological complexes. In the past few years, the most prolific branch of cryo-EM, single particle analysis, has revolutionized the structural biology field and structural investigation of membrane proteins, in particular. To achieve high-resolution structures of macromolecules in noncrystalline specimens, from sample and grid preparation to image acquisition, image data processing, and analysis of 3D maps, methodological advances in each of the steps play critical roles. In this Review, we discuss two areas in single particle cryo-EM, the remarkable developments in sample preparation strategies, particularly for membrane proteins, and breakthroughs in methodologies for molecular model building on the high-resolution 3D density maps that brought promises to resolve high-resolution (<4 Å) structures of biological macromolecules.

中文翻译：

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单粒子低温电子显微镜的回顾与展望：以整体膜蛋白一类为例。

低温电子显微镜（cryo-EM）领域的巨大技术飞跃确保了生物大分子的近原子分辨率结构的实现。为了表彰这项成就，2017年诺贝尔化学奖被授予了低温电磁领域的先驱雅克·杜博谢特，约阿希姆·弗兰克和理查德·亨德森。目前，该技术已成为异质和内在动态生物复合物结构分析的首选方法。在过去的几年中，冷冻EM的最多产分支，即单颗粒分析，彻底改变了结构生物学领域，尤其是膜蛋白的结构研究。为了实现非晶体标本中大分子的高分辨率结构，从样品和网格制备到图像采集，图像数据处理以及3D地图分析，每个步骤中的方法进步都起着至关重要的作用。在这篇综述中，我们讨论了单粒子冷冻EM的两个领域，样品制备策略的显着进展（尤其是膜蛋白）以及高分辨率3D密度图上分子模型构建方法的突破，这带来了解决高分离度的希望。大分子的高分辨率（<4Å）结构。