Ribosome structures to near-atomic resolution from thirty thousand cryo-EM particles.
Ribosome structures to near-atomic resolution from thirty thousand cryo-EM particles.
复制标题
DOI：
10.7554/elife.00461
复制
发表时间：
2013-02-19
期刊：
影响因子：
7.7
通讯作者：
Scheres SH
中科院分区：
文献类型：
--
作者：
Bai XC;Fernandez IS;McMullan G;Scheres SH
尽管低温电子显微镜（cryo-EM）单粒子分析已成为大型柔性大分子组装体结构生物学的重要工具，但该技术尚未充分发挥其潜力。除了辐射损伤造成的基本限制外，不良的探测器和光束引起的样品移动也会降低可达到的分辨率。新一代的直接电子探测器可能会改善这两种影响。除了表现出改善的信噪比性能外，这些相机还足够快，可以在电子辐照期间跟踪粒子运动。在这里，我们评估这种技术的潜力，用于冷冻EM结构测定。使用一种新开发的统计电影处理方法来补偿光束引起的运动，我们表明，核糖体重建与前所未有的分辨率可以计算从几乎两个数量级的粒子比以前使用的。因此，这种方法可以将高分辨率cryo-EM的范围扩展到广泛的生物标本。http://dx.doi.org/10.7554/eLife.00461.001在原子水平上确定蛋白质和其他生物分子的结构是理解生物学许多方面的核心。X射线晶体学是结构生物学中最著名的技术，但顾名思义，它只适用于可以结晶的样品。电子冷冻显微镜（cryo-EM）可能用于确定无法结晶的生物分子的原子结构，但目前这种方法可以实现的分辨率仅足以成像某些类型的病毒。在cryo-EM中，感兴趣的生物分子的溶液被冷冻在一层薄薄的冰中，并且该层在电子显微镜中成像。通过组合许多不同方向的许多相同生物分子的图像，可以向后工作并确定它们的3D结构。然而，为了以高分辨率确定这种结构，有必要进行重复测量以减少图像中的高水平噪声。冷冻EM图像通常记录在照相胶片或CCD（电荷耦合器件）相机上。然而，照相胶片不适合高通量方法，因为它必须手动处理，而CCD相机的效率有限，因为电子必须转换为可见光才能被检测到。可以直接检测电子的数码相机最近已经上市，并且比胶片和CCD相机更有效。它们的速度也快得多，这意味着可以在样品暴露于电子束的时间（通常为100秒）内记录样品的视频。理论上，处理这些视频可以补偿电子束引起的生物分子的任何运动。沿着由电子引起的辐射损伤，这些射束诱导的运动已经成为对利用低温EM可以实现的分辨率的主要限制。Bai等人通过确定两个核糖体的结构证明了直接电子探测器在冷冻EM中的潜力。使用一种新的统计算法来精确地跟踪核糖体在暴露于电子束期间的运动，它们能够补偿这些运动，这使得可以以接近原子的精度确定核糖体的结构。此外，他们仅用200，000个核糖体就实现了比以前用超过一百万个核糖体实现的分辨率更好的分辨率，允许核糖体内部的小细节-例如β-链和庞大的氨基酸侧链-首次用cryo-EM解析。因此，Bai等人的工作可以让研究人员使用冷冻EM以原子精度确定更多生物分子的结构。DOI：http://dx.doi.org/10.7554/eLife.00461.002网站
Although electron cryo-microscopy (cryo-EM) single-particle analysis has become an important tool for structural biology of large and flexible macro-molecular assemblies, the technique has not yet reached its full potential. Besides fundamental limits imposed by radiation damage, poor detectors and beam-induced sample movement have been shown to degrade attainable resolutions. A new generation of direct electron detectors may ameliorate both effects. Apart from exhibiting improved signal-to-noise performance, these cameras are also fast enough to follow particle movements during electron irradiation. Here, we assess the potentials of this technology for cryo-EM structure determination. Using a newly developed statistical movie processing approach to compensate for beam-induced movement, we show that ribosome reconstructions with unprecedented resolutions may be calculated from almost two orders of magnitude fewer particles than used previously. Therefore, this methodology may expand the scope of high-resolution cryo-EM to a broad range of biological specimens. DOI: http://dx.doi.org/10.7554/eLife.00461.001 Determining the structure of proteins and other biomolecules at the atomic level is central to understanding many aspects of biology. X-ray crystallography is the best-known technique for structural biology but, as the name suggests, it works only with samples that can be crystallized. Electron cryo-microscopy (cryo-EM) could, potentially, be used to determine the atomic structures of biomolecules that cannot be crystallized, but at present the resolution that can be achieved with this approach is sufficient only for imaging certain types of viruses. In cryo-EM, a solution of the biomolecule of interest is frozen in a thin layer of ice, and this layer is imaged in an electron microscope. By combining images of many identical biomolecules in many different orientations, it is possible to work backwards and determine their 3D structure. However, in order to determine this structure at high resolution, it is necessary to make repeated measurements to reduce high levels of noise in the images. Cryo-EM images are usually recorded on a photographic film or a CCD (charge-coupled device) camera. However, photographic film is unsuitable for high-throughput methods because it has to be handled manually, while the efficiency of CCD cameras is limited because the electrons have to be converted into visible light to be detected. Digital cameras that can detect electrons directly have become available recently, and these are more efficient than both film and CCD cameras. They are also much faster, which means that it is possible to record videos of the sample during the time (typically ∼1 s) it is being exposed to the electron beam. Processing these videos could then—in theory—compensate for any movements of the biomolecules that are induced by the electron beam. Along with radiation damage caused by the electrons, these beam-induced movements have been a major limitation on the resolution that can be achieved with cryo-EM. Bai et al. demonstrate the potential of direct-electron detectors in cryo-EM by determining the structures of two ribosomes. Using a novel statistical algorithm to accurately follow the movements of the ribosomes during the time they are exposed to the electron beam, they are able to compensate for these movements, and this makes it possible to determine the structures of the ribosomes with near-atomic precision. Moreover, the resolution they achieve with just ∼30,000 ribosomes is better than that previously achieved with more than a million ribosomes, allowing small details inside the ribosome – such as ß-strands and bulky amino-acid side chains – to be resolved with cryo-EM for the first time. The work of Bai et al. could, therefore, allow researchers to use cryo-EM to determine the structure of many more biomolecules with atomic precision. DOI: http://dx.doi.org/10.7554/eLife.00461.002