Sub-3 Å resolution protein structure determination by single-particle cryo-EM at 100 keV

Dimple Karia, Adrian F Koh, Wen Yang, Victoria I Cushing, Benjamin Basanta, Daniel B Mihaylov, Sagar Khavnekar, Ondřej Vyroubal, Miloš Malínský, Ondřej Sháněl, Vojtëch Doležal, Juergen M Plitzko, Lingbo Yu, Gabriel C Lander, A. Radu C Aricescu, Basil J Greber, Abhay Kotecha
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Abstract

Cryo-electron microscopy (cryo-EM) has revolutionized structural biology by providing high-resolution insights into biological macromolecules. Here, we present sub-3 Å resolution structures determined using the 100 keV Tundra cryogenic transmission electron microscope (cryo-TEM), equipped with the newly developed Falcon C direct electron detector (DED). Our results demonstrate that this lower voltage microscope, when combined with advanced electron optics and detectors, can achieve high-resolution reconstructions that were previously only attainable with higher voltage systems. The implementation of an extreme-brightness field emission gun (XFEG) and an SP-TWIN objective lens significantly enhanced the spatial and temporal coherence of the system. Furthermore, the semi-automated sample loader minimized contamination and drift, allowing extended data collection sessions without manual intervention. The high detective quantum efficiency (DQE) of Falcon C further improved the signal-to-noise ratio, which is critical for achieving high-resolution structures. We validated the performance of this microscope by determining the structures of various biological samples, including apoferritin, T20S proteasome, GABAA receptor, haemoglobin, and human transthyretin ranging in size from 440 kDa to 50 kDa. The highest resolutions achieved were 2.1 Å for apoferritin, 2.7 Å for the 20S proteasome, 2.8 Å for the GABAA receptor, 5.0 Å for haemoglobin, and 3.5 Å for transthyretin. We also explored a larger specimen, a 3.9 MDa Adeno-associated virus (AAV9) capsid and resolved it a 2.8 Å. This work highlights the potential of 100 keV TEMs to make high-resolution cryo-EM more accessible to the structural biology community. Furthermore, it sets a precedent for the use of lower voltage TEMs in routine cryo-EM studies, not only for screening grids for single particle analysis but also for achieving high-resolution structures of protein samples.
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在 100 keV 下通过单粒子低温电子显微镜确定 3 Å 以下分辨率的蛋白质结构
低温电子显微镜(cryo-EM)通过提供高分辨率的生物大分子洞察力,彻底改变了结构生物学。在这里,我们展示了使用 100 keV Tundra 低温透射电子显微镜(冷冻电子显微镜)测定的亚 3 Å 分辨率结构,该显微镜配备了新开发的 Falcon C 直接电子探测器(DED)。我们的研究结果表明,这种低电压显微镜与先进的电子光学和探测器相结合,可以实现高分辨率重构,而这在以前只能通过更高的电压系统才能实现。极亮度场发射枪(XFEG)和 SP-TWIN 物镜的应用大大提高了系统的空间和时间一致性。此外,半自动样品装载器最大限度地减少了污染和漂移,无需人工干预即可进行长时间的数据采集。Falcon C 的高探测量子效率(DQE)进一步提高了信噪比,这对实现高分辨率结构至关重要。我们通过测定各种生物样本的结构验证了这台显微镜的性能,包括从 440 kDa 到 50 kDa 大小不等的载脂蛋白、T20S 蛋白酶体、GABAA 受体、血红蛋白和人类转甲状腺素。所达到的最高分辨率分别为:apoferritin 2.1 Å、20S 蛋白酶体 2.7 Å、GABAA 受体 2.8 Å、血红蛋白 5.0 Å 和转甲状腺素 3.5 Å。我们还研究了一个更大的样本,即 3.9 MDa 的腺相关病毒 (AAV9) 荚膜,并将其解析为 2.8 Å。这项工作突出了 100 keV TEM 在使结构生物学界更容易获得高分辨率冷冻电镜方面的潜力。此外,它还开创了在常规冷冻电镜研究中使用低电压 TEM 的先例,不仅可用于筛选网格进行单颗粒分析,还可用于实现蛋白质样本的高分辨率结构。
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