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Measuring the effects of particle orientation to improve the efficiency of electron cryomicroscopy.

ABSTRACT: The orientation distribution of a single-particle electron cryomicroscopy specimen limits the resolution of the reconstructed density map. Here we define a statistical quantity, the efficiency, E od, which characterises the orientation distribution via its corresponding point spread function. The efficiency measures the ability of the distribution to provide uniform information and resolution in all directions of the reconstruction, independent of other factors. This metric allows rapid and rigorous evaluation of specimen preparation methods, assisting structure determination to high resolution with minimal data.A number of parameters influence the resolution of a cryo-EM structure. Here the authors investigate the effects of specimen orientation in single particle cryo-EM and present open-source software for rapidly assessing orientation distributions to improve data collection.

SUBMITTER: Naydenova K

PROVIDER: S-EPMC5607000 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Measuring the effects of particle orientation to improve the efficiency of electron cryomicroscopy.

Naydenova Katerina K Russo Christopher J CJ

Nature communications 20170920 1

The orientation distribution of a single-particle electron cryomicroscopy specimen limits the resolution of the reconstructed density map. Here we define a statistical quantity, the efficiency, E <sub>od</sub>, which characterises the orientation distribution via its corresponding point spread function. The efficiency measures the ability of the distribution to provide uniform information and resolution in all directions of the reconstruction, independent of other factors. This metric allows rap ...[more]

PMID: 28931821

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Project description: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).

Dataset Information

Measuring the effects of particle orientation to improve the efficiency of electron cryomicroscopy.

Publications

Measuring the effects of particle orientation to improve the efficiency of electron cryomicroscopy.

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