Reliable cryo-EM resolution estimation with modified Fourier shell correlation.

Pawel A Penczek

doi:10.1107/s2052252520011574

Abstract

A modified Fourier shell correlation (mFSC) methodology is introduced that is aimed at addressing two fundamental problems that mar the use of the FSC: the strong influence of mask-induced artifacts on resolution estimation and the lack of assessment of FSC uncertainties stemming from the inability to determine the associated number of degrees of freedom. It is shown that by simply changing the order of the steps in which the FSC is computed, the correlations induced by masking of the input data can be eliminated. In addition, to further reduce artifacts, a smooth Gaussian window function is used to outline the regions of reciprocal space within which the mFSC is computed. Next, it is shown that the number of degrees of freedom (ndf) of the system is approximated well by combining the ndf associated with the Gaussian window in reciprocal space with further reduction of the ndf owing to the use of the mask in real space. It is demonstrated through the application of the mFSC to both single-particle and helical structures that the mFSC yields reliable, mask-induced artifact-free results as a result of the introduced modifications. Since the adverse effect of the mask is eliminated, it also becomes possible to compute robust local resolutions both per voxel of a 3D map as well as, in a newly developed approach, per functional subunit, segment or even larger secondary element of the studied complex.

Highlights

In single-particle reconstructions, it is essential to have a robust measure of the reliability of the resulting 3D structure derived from 2D cryo-EM projection images
For tests of single-particle resolution estimation by the modified Fourier shell correlation (FSC) (mFSC), we used the data set for the Plasmodium falciparum 80S ribosome bound to the antiprotozoan drug emetine deposited in the EMPIAR archive as entry EMPIAR-10028
We have addressed two fundamental issues in resolution estimation of cryo-EM maps using the FSC methodology: (i) the strong influence of a mask on the results and (ii) the estimation of the number of degrees of freedom in the data, which allowed us to properly calculate one-sided confidence intervals (CIs) for FSC coefficients

Summary

Introduction

In single-particle reconstructions, it is essential to have a robust measure of the reliability of the resulting 3D structure derived from 2D cryo-EM projection images. It can be argued that the shape of the entire FSC/SSNR curve is an indicator of the structure quality. Such a number is determined from the FSC curve as the spatial frequency at which the curve drops below a predefined threshold and is called the resolution (and is typically given in units of inverted spatial frequency, length). It is apparent that two different structures can have the same resolution but quite different quality, as the latter is given by all FSC coefficients, not just one point that might be shared between two different results (Penczek, 2010)

Methods

Results

Conclusion