Most high-resolution information loss in cryomicrographs stems from radiation damage and particle movement in the vitrified specimen during imaging. Recently, we elucidated the physical origins of cryoEM specimen movement and demonstrated a simple strategy for eliminating it. Movement-free imaging not only increases the information content of cryoEM data, but also allows for a new approach to 3D reconstruction from averaged 2D images: zero-dose extrapolation, similar to a previously proposed approach in X-ray crystallography. We describe an algorithm for zero-dose extrapolation, which replaces image-wighting for movement-free cryoEM data, and demonstrate its implementation and use in a standalone program, Zero Dose. We show how structure determination at zero dose has allowed us allowed us to better visualise ligand binding, metal coordination, solvent molecules, alternative side chain conformations, lipid bilayers and other important structural features of various specimens. In addition, this approach to cryoEM reconstruction allows for new insights into radiation damage of biological molecules during imaging with high-energy electrons, and facilitates atomic model refinement.
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