Accurate modeling of single-particle cryo-EM images quantitates the benefits expected from using Zernike phase contrast

Abstract

The use of a Zernike-type phase plate in biological cryo-electron microscopy allows the imaging, without using defocus, of what are predominantly phase objects. It is thought that such phase-plate implementations might result in higher quality images, free from the problems of CTF correction that occur when images must be recorded at extremely high values of defocus. In single-particle cryo-electron microscopy it is hoped that these improvements in image quality will facilitate work on structures that have proved difficult to study, either because of their relatively small size or because the structures are not completely homogeneous. There is still a need, however, to quantify how much improvement can be gained by using a phase plate for single-particle cryo-electron microscopy. We present a method for quantitatively modelling the images recorded with 200 keV electrons, for single particles embedded in vitreous ice. We then investigate what difference the use of a phase-plate device could have on the processing of single-particle data. We confirm that using a phase plate results in single-particle datasets in which smaller molecules can be detected, particles can be more accurately aligned and problems of heterogeneity can be more easily addressed.

$\gamma$-SUP: A clustering algorithm for cryo-electron microscopy images of asymmetric particles

“…Gaussian noise N (0, σ 2 ε ) added, where σ ε = 40, 50, 60, so that the corresponding SNRs are 0.19, 0.12, 0.08, which reflect the low SNR nature of cryo-EM images. This procedure of simulating cryo-EM images is commonly used in the cryo-EM community [Chang et al (2010), Singer et al (2010), Sorzano et al (2010), Hall, Nogales and Glaeser (2011)]. To reflect the nature of the experiment, we consider two scenarios:…”

Methods for testing Zernike phase plates and a report on silicon-based phase plates with reduced charging and improved ageing characteristics

“…The usefulness of in-focus TEM phase-plate imaging has long been appreciated, and has recently been affirmed (Malac et al, 2008; Chang et al, 2010; Hall et al, 2011), but practical problems with its implementation for “real-world” use have hindered widespread adoption (see reviews by Nagayama and Danev, 2009; Danev et al, 2009; Danev and Nagayama, 2010; Nagayama 2011). Nevertheless, the current state of development has enabled early applications to biological problems, validating the technical advantages of phase-plate imaging (Danev and Nagayama, 2008; Fukuda and Nagayama, 2012; Rochat et al, 2011; Hosogi et al, 2011; Danev et al, 2010; Murata et al, 2010; Shigematsu et al, 2010; Yui et al, 2008; Yamaguchi et al, 2008).…”

Hydration-layer models for cryo-EM image simulation

“…A recent example is the use of a quantitative model of image formation to evaluate the potential of phase-contrast electron microscopy (Hall et al, 2011). Traditionally such simulations start with the placement of electron-scattering density according to the atomic coordinates of a protein molecule.…”

“…One approach to modeling protein solvation is to compute directly the average density and distribution of water molecules surrounding a protein by molecular dynamics (MD) simulation (Wang et al, 2008; Hall et al, 2011). Such calculations are however very time consuming.…”