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首页> 外文期刊>Journal of Physics, D. Applied Physics: A Europhysics Journal >Zernike phase contrast cryo-electron microscopy reveals 100 kDa component in a protein complex
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Zernike phase contrast cryo-electron microscopy reveals 100 kDa component in a protein complex

机译：Zernike相衬低温电子显微镜揭示蛋白质复合物中的100 kDa成分

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Cryo-electron microscopy (cryo-EM) has become a powerful technique for obtaining near atomic structures for large protein assemblies or large virus particles, but the application to protein particles smaller than 200-300 kDa has been hampered by the feeble phase contrast obtained for such small samples and the limited number of electrons tolerated by them without incurring excessive radiation damage. By implementing a thin-film quarter-wave phase plate to a cryo-EM, Nagayama, one of the present authors, has recently restored the long-lost very low spatial frequencies, generating in-focus phase contrast superior to that of conventional defocusing phase contrast, and successfully applied the so-called Zernike phase-plate cryo-EM to target various biological samples in native state. Nevertheless, the sought-after goal of using enhanced phase contrast to reveal a native protein as small as 100 kDa waits to be realized. Here, we report a study in which 200 kV Zernike phase-plate cryo-EM with a plate cut-on periodicity of 36 nm was applied to visualize 100 kDa components of various protein complexes, including the small domains on the surface of an icosahedral particle of ～38 nm derived from the dragon grouper nervous necrosis virus (DGNNV) and the labile sub-complex dissociated from yeast RNA polymerase III of 17 nm. In the former case, we observed a phase contrast reversal phenomenon at the centre of the icosahedral particle and traced its root cause to the near matching of the cut-on size and the particle size. In summary, our work has demonstrated that Zernike phase-plate implementation can indeed expand the size range of proteins that can be successfully investigated by cryo-EM, opening the door for countless proteins. Finally, we briefly discuss the possibility of using a transfer lens system to enlarge the cut-on periodicity without further miniaturizing the plate pinhole.
机译：低温电子显微镜（cryo-EM）已成为一种强大的技术，可用于获取大型蛋白质装配体或大型病毒颗粒的近原子结构，但对于小于200-300 kDa的蛋白质颗粒，其获得的微弱相衬阻碍了其应用。如此小的样品以及它们所能承受的有限数量的电子，而不会造成过度的辐射损伤。通过在低温EM上安装薄膜四分之一波相位板，长谷山（目前的作者之一）最近恢复了长期丢失的非常低的空间频率，产生的聚焦相位对比度优于传统的散焦相位。与此相反，成功地将所谓的Zernike相板式cryo-EM用于天然状态的各种生物样品。然而，使用增强的相衬来揭示小至100 kDa的天然蛋白质的抢手目标有待实现。在这里，我们报道了一项研究，其中采用200 kV Zernike相板冷冻-EM，板切开周期性为36 nm，以可视化各种蛋白质复合物的100 kDa成分，包括二十面体颗粒表面的小区域来源于龙石斑鱼神经坏死病毒（DGNNV）的〜38 nm和从17 nm酵母RNA聚合酶III解离的不稳定亚复合物。在前一种情况下，我们在二十面体颗粒的中心观察到相衬反转现象，并将其根本原因追溯到截留尺寸和颗粒尺寸的接近匹配上。总而言之，我们的工作表明Zernike相板的实施确实可以扩大蛋白质的大小范围，可以通过cryo-EM成功进行研究，从而为无数蛋白质打开了大门。最后，我们简要讨论了使用转移透镜系统扩大切角周期而不进一步缩小印版针孔的可能性。

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