Abstract
Single particle cryogenic electron microscopy (cryo-EM) is transforming structural biology by enabling the analysis of difficult macromolecular specimens, such as membrane proteins or large complexes with flexible elements, at near atomic resolution with an accuracy close to that of X-ray crystallography. As the technique continues to improve, it is important to assess and exploit its full potential to produce the most possible reliable atomic models. Here we propose to use the experimental images as the data for refinement and validation, instead of the reconstructed maps as currently used. This procedure, which is in spirit quite similar to that used in X-ray crystallography where the data include experimental phases, should contribute to improve the quality of the cryo-EM atomic models.
Highlights
Single particle cryogenic electron microscopy is transforming structural biology by enabling the analysis of difficult macromolecular specimens, such as membrane proteins or large complexes with flexible elements, at near atomic resolution with an accuracy close to that of X-ray crystallography
The technique presents a clear advantage over X-ray crystallography in that it allows the structural analysis of particles that are difficult or impossible to grow into crystals, such as very large complexes, membrane proteins or proteins with flexibles portions, and is gaining a prevalent role in the determination of biological macromolecular structures[1]
A recent comparison of X-ray and cryogenic electron microscopy (cryo-EM) maps calculated at the same resolution, together with the corresponding atomic models, showed that the appearance of the maps was quite comparable between the two techniques, X-ray crystallography maps were more detailed and the atomic models fitted into them were more accurate[4]
Summary
By using experimental images as data, cryo-EM refinement procedures become quite similar to those used in X-ray crystallography, in spirit, in that the reconstructed maps are allowed to improve as model building and refinement proceed. Other not well understood aspects throughout the determination procedures will need to be improved, such as dealing with errors on the detectors or multiple conformations of molecules[6], some of which may benefit from the proposed refinement target to yield statistically more robust structures This may allow to better exploit the potential of the cryo-EM method and lead to a significant gain of accuracy of the high-resolution refinement protocol.
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