Description and comparison of algorithms for correcting anisotropic magnification in cryo-EM images

TL;DR

This paper compares different algorithms for correcting anisotropic magnification in cryo-EM images, addressing a key source of distortion that affects 3D structure determination.

Contribution

It introduces and evaluates real space and Fourier space methods for correcting anisotropic magnification in cryo-EM images, and proposes a way to adjust CTF parameters affected by this distortion.

Findings

Four different correction methods are described and compared.

Anisotropic magnification causes systematic errors in CTF parameter estimation.

Detecting anisotropy can be achieved by analyzing CTF parameters from distorted images.

Abstract

Single particle electron cryomicroscopy (cryo-EM) allows for structures of proteins and protein complexes to be determined from images of non-crystalline specimens. Cryo-EM data analysis requires electron microscope images of randomly oriented ice-embedded protein particles to be rotated and translated to allow for coherent averaging when calculating three-dimensional (3D) structures. Rotation of 2D images is usually done with the assumption that the magnification of the electron microscope is the same in all directions. However, due to electron optical aberrations, this condition is not met with some electron microscopes when used with the settings necessary for cryo-EM with a direct detector device (DDD) camera. Correction of images by linear interpolation in real space has allowed high-resolution structures to be calculated from cryo-EM images for symmetric particles. Here we describe and compare a simple real space method, a simple Fourier space method, and a somewhat more sophisticated Fourier space method to correct images for a measured anisotropy in magnification. Further, anisotropic magnification causes contrast transfer function (CTF) parameters estimated from image power spectra to have an apparent systematic astigmatism. To address this problem we develop an approach to adjust CTF parameters measured from distorted images so that they can be used with corrected images. The effect of anisotropic magnification on CTF parameters provides a simple way of detecting magnification anisotropy in cryo-EM datasets.