Prevention of overfitting in cryo-em structure determination

Access through your institution Buy or subscribe To the Editor: In the field of single-particle analysis of electron cryomicroscopy (cryo-EM) data, a growing concern that some resolution

claims might not be substantiated by the data has helped instigate community-wide efforts to develop new validation tools1. A known issue with commonly used cryo-EM structure determination

procedures is their tendency to overfit the data. Most procedures counter overfitting by low-pass filtering, but the effective frequencies for these filters are often based on suboptimal

Fourier shell correlation2 (FSC) procedures. In the suboptimal procedure, FSC curves are calculated between reconstructions from two halves of the data, and a single model is used to

determine the relative orientations of all particles. It is well known that bias toward noise in the single model may inflate the resulting resolution estimates. To illustrate this, we

applied the suboptimal procedure to a simulated cryo-EM data set of 20,212 GroEL particles. Whereas the reported resolution was 4.6 Å, the true resolution of the map was only 7.8 Å. Also,

the presence of expected density features in the map does not necessarily provide sufficient evidence for a resolution claim: we made convincing figures of apparent side-chain density that

in reality corresponded to overfitted noise (Supplementary Fig. 1). Consequently, overfitting may remain undetected, and interpretation of cryo-EM maps may be subject to errors. This is a

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during checkout ADDITIONAL ACCESS OPTIONS: * Log in * Learn about institutional subscriptions * Read our FAQs * Contact customer support REFERENCES * Henderson, R. et al. _Structure_ 20,

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ACKNOWLEDGEMENTS We are grateful to T. Crowther and R. Henderson for helpful discussions and to J. Grimmett for help with computing. T. Crowther provided hepatitis B data, and the National

Center for Macromolecular Imaging, which is funded by US National Institutes of Health grant P41RR02250, provided GroEL data. This work was funded by the UK Medical Research Council through

grant MC_UP_A025_1013 to S.H.W.S. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Medical Research Council Laboratory of Molecular Biology, Cambridge, UK Sjors H W Scheres & Shaoxia Chen

Authors * Sjors H W Scheres View author publications You can also search for this author inPubMed Google Scholar * Shaoxia Chen View author publications You can also search for this author

inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Sjors H W Scheres. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. SUPPLEMENTARY

INFORMATION SUPPLEMENTARY TEXT AND FIGURES Supplementary Figures 1–4, Supplementary Table 1, Supplementary Methods and Supplementary Software (PDF 765 kb) RIGHTS AND PERMISSIONS Reprints and

permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Scheres, S., Chen, S. Prevention of overfitting in cryo-EM structure determination. _Nat Methods_ 9, 853–854 (2012).

https://doi.org/10.1038/nmeth.2115 Download citation * Published: 29 July 2012 * Issue Date: September 2012 * DOI: https://doi.org/10.1038/nmeth.2115 SHARE THIS ARTICLE Anyone you share the

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