High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy

By Shaoxia Chen, G. McMullan, A. R. Faruqi, G. Murshudov, J. Short, Sjors H. W. Scheres, R. Henderson. 2013

984 citations recorded by the source corpus.

Three-dimensional (3D) structure determination by single particle electron cryomicroscopy (cryoEM) involves the calculation of an initial 3D model, followed by extensive iterative improvement of the orientation determination of the individual particle images and the resulting 3D map. Because there is much more noise than signal at high resolution in the images, this creates the possibility of noise reinforcement in the 3D map, which can give a false impression of the resolution attained. The balance between signal and noise in the final map at its limiting resolution depends on the image processing procedure and is not easily predicted. There is a growing awareness in the cryoEM community of how to avoid such over-fitting and over-estimation of resolution. Equally, there has been a reluctance to use the two principal methods of avoidance because they give lower resolution estimates, which some people believe are too pessimistic. Here we describe a simple test that is compatible with any image processing protocol. The test allows measurement of the amount of signal and the amount of noise from overfitting that is present in the final 3D map. We have applied the method to two different sets of cryoEM images of the enzyme beta-galactosidase using several image processing packages. Our procedure involves substituting the Fourier components of the initial particle image stack beyond a chosen resolution by either the Fourier components from an adjacent area of background, or by simple randomisation of the phases of the particle structure factors. This substituted noise thus has the same spectral power distribution as the original data. Comparison of the Fourier Shell Correlation (FSC) plots from the 3D map obtained using the experimental data with that from the same data with high-resolution noise (HR-noise) substituted allows an unambiguous measurement of the amount of overfitting and an accompanying resolution assessment. A simple formula can be used to calculate an unbiased FSC from the two curves, even when a substantial amount of overfitting is present. The approach is software independent. The user is therefore completely free to use any established method or novel combination of methods, provided the HR-noise test is carried out in parallel. Applying this procedure to cryoEM images of beta-galactosidase shows how overfitting varies greatly depending on the procedure, but in the best case shows no overfitting and a resolution of ~6 Å. (382 words)

Subfields: Molecular Biology, Structural Biology, Surfaces, Coatings and Films

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Citations in the corpus

Induction of Potent Neutralizing Antibody Responses by a Designed Protein Nanoparticle Vaccine for Respiratory Syncytial Virus (2019, methodology): “143 criterion (Rosenthal and Henderson, 2003) and Fourier shell correlation curves were corrected for the effects of soft masking by high-resolution noise substitution (Chen et al., 2013).”
Helical reconstruction in RELION (2016, positive): “…RELION's post-processing job-type, we then applied automated B-factor sharpening (Rosenthal and Henderson, 2003) and corrected the FSC between the two independently refined maps of the 3D auto-refinement for the convolution effects of a solvent mask using phase randomisation (Chen et al., 2013).”
Visualization of two architectures in class-II CAP-dependent transcription activation (2019, methodology): “29 Å resolution for the map outputted from Auto refinement [38].”
New tools for the analysis and validation of cryo-EM maps and atomic models (2018, methodology): “…(Rosenthal & Henderson, 2003; Heymann & Belnap, 2007; Penczek, 2010; Murshudov & Evans, 2012; Karplus & Diederichs, 2012; Urzhumtseva et al., 2013, Chen et al., 2013; Kucukelbir et al., 2014; see also web service provided by GlobalPhasing2), there is still debate about the appropriate…”
Glycan Shield and Fusion Activation of a Deltacoronavirus Spike Glycoprotein Fine-Tuned for Enteric Infections (2017, neutral): “soft masking by high-resolution noise substitution(72).”
Decoding Mammalian Ribosome-mRNA States by Translational GTPase Complexes (2016, methodology): “High-resolution noise substitution was used to correct for the effects of a soft mask on FSC curves (Chen et al., 2013).”