Zernike Phase Contrast Cryo-Electron Microscopy and Tomography for Structure Determination at Nanometer and Subnanometer Resolutions

National Center for Macromolecular Imaging, Verna and Marrs Mclean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA.
Structure (Impact Factor: 5.62). 08/2010; 18(8):903-12. DOI: 10.1016/j.str.2010.06.006
Source: PubMed


Zernike phase contrast cryo-electron microscopy (ZPC-cryoEM) is an emerging technique that is capable of producing higher image contrast than conventional cryoEM. By combining this technique with advanced image processing methods, we achieved subnanometer resolution for two biological specimens: 2D bacteriorhodopsin crystal and epsilon15 bacteriophage. For an asymmetric reconstruction of epsilon15 bacteriophage, ZPC-cryoEM can reduce the required amount of data by a factor of approximately 3, compared with conventional cryoEM. The reconstruction was carried out to 13 A resolution without the need to correct the contrast transfer function. New structural features at the portal vertex of the epsilon15 bacteriophage are revealed in this reconstruction. Using ZPC cryo-electron tomography (ZPC-cryoET), a similar level of data reduction and higher resolution structures of epsilon15 bacteriophage can be obtained relative to conventional cryoET. These results show quantitatively the benefits of ZPC-cryoEM and ZPC-cryoET for structural determinations of macromolecular machines at nanometer and subnanometer resolutions.

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    • "Glaeser et al. (2013) showed that, with phase plate, small proteins such as individual streptavidin tetramers can be clearly distinguished in cryo. Furthermore, using phase contrast cryoEM, the 3D structure of epsilon15 bacteriophage can be resolved at nanometer resolution even with no symmetry imposed (Murata et al., 2010). Remarkably, structural details such as the tail and capsid of an individual phage can be directly delineated by electron cryo-tomographic reconstruction (Murata et al., 2010). "
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