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Free keywords:
TRANSMISSION ELECTRON-MICROSCOPY; HUMAN 80S RIBOSOME; CRYOELECTRON
MICROSCOPY; CONTRAST; VISUALIZATION; TOMOGRAPHY; REFINEMENT; COMPLEX;
DESIGN; SYSTEMBiochemistry & Molecular Biology; Biophysics; Cell Biology; Cryo electron microscopy; Volta phase plate; Image processing; Ribosome;
High-resolution cryo-EM; Structural biology;
Abstract:
A current bottleneck in structure determination of macromolecular complexes by cryo electron microscopy (cryo-EM) is the large amount of data needed to obtain high-resolution 3D reconstructions, including through sorting into different conformations and compositions with advanced image processing. Additionally, it may be difficult to visualize small ligands that bind in sub-stoichiometric levels. Volta phase plates (VPP) introduce a phase shift in the contrast transfer and drastically increase the contrast of the recorded low-dose cryo-EM images while preserving high frequency information. Here we present a comparative study to address the behavior of different data sets during image processing and quantify important parameters during structure refinement. The automated data collection was done from the same human ribosome sample either as a conventional defocus range dataset or with a Volta phase plate close to focus (cfVPP) or with a small defocus (dfVPP). The analysis of image processing parameters shows that dfVPP data behave more robustly during cryo-EM structure refinement because particle alignments, Euler angle assignments and 2D & 3D classifications behave more stably and converge faster. In particular, less particle images are required to reach the same resolution in the 3D reconstructions. Finally, we find that defocus range data collection is also applicable to VPP. This study shows that data processing and cryo-EM map interpretation, including atomic model refinement, are facilitated significantly by performing VPP cryo-EM, which will have an important impact on structural biology.
