LAUSR

Cryo-electron microscopy (cryo-EM) single particle analysis has come a long way in achieving atomic-level resolution when imaging biomolecules. In order to obtain the best possible three-dimensional structure in cryo-EM, many parameters have to be carefully considered. Here we address the often-overlooked parameter of the pixel size, which describes the magnification of the image produced by the experiment. While efforts are made to refine and validate this parameter in the analysis of cryo-EM experimental data, there is no systematic protocol in place. Since the pixel size parameter can have an impact on the resolution and accuracy of a cryo-EM map, and the atomic resolution, three-dimensional structure models derived from it, we propose a computational protocol to estimate the appropriate pixel size parameter. In our protocol, we fit and refine atomic structures against cryo-EM map at multiple pixel sizes. The resulting fitted and refined structures are evaluated using the GOAP (Generalized Orientation-Dependent, All-atom Statistical Potential) score, which we found to perform better than other commonly used functions, such as Molprobity and the correlation coefficient from refinement. Finally, we describe the efficacy of this protocol in retrieving appropriate pixel sizes for several examples; simulated data based on yeast elongation factor 2, and experimental data from Gro-EL chaperone, beta-galactosidase and the TRPV1 ion channel.