Radostin Danev

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

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.

Zernike phase contrast electron microscopy of ice-embedded influenza A virus.

The ultrastructure of the frozen-hydrated influenza A virus was examined by Zernike phase contrast electron microscopy. Using this new microscopy, not only lipid bilayers but also individual glycoprotein spikes on viral envelopes were clearly resolved with high contrast in micrographs taken in focus. In addition to spherical and elongated virions, three other classes of virions were distinguished on the basis of the features of their viral envelope: virions with a complete matrix layer, which were the most predominant, virions with a partial matrix layer, and virions with no matrix layer under the lipid bilayer. About 450 glycoprotein spikes were present in an average-sized spherical virion. Eight ribonucleoprotein complexes, that is, a central one surrounded by seven others, were distinguished in one viral particle. Thus, Zernike phase contrast electron microscopy is a powerful tool for resolving the ultrastructure of viruses, because it enables high-contrast images of ice-embedded particles free of contrast transfer function artifacts that can be a problem in conventional cryo-electron microscopy.