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bioRxiv · 10.1101/2025.11.26.690836

Preserving Information and Integrity in Cryo-EM: A Fourier-Space Deposition Approach

Otwinowski, Z.·Bromberg, R.·Guo, Y.·Borek, D.
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Abstract

Current guidelines for depositing cryogenic electron microscopy single particle reconstruction (cryo-EM SPR) data require submission of unfiltered, unmasked, and unsharpened raw half-maps. The Fourier Shell Correlation (FSC) between the half-maps is then used as a proxy for the signal-to-noise ratio (SNR) to estimate the reconstructions resolution. This policy was introduced to enable independent validation of reported resolutions. Although developed to safeguard data integrity and minimize bias, these guidelines do not account for specific features of modern cryo-EM processing software, in particular weighting schemes that are not retained in half-map depositions and yet in general are necessary to recapitulate resolution estimates. As a results, resolution estimates and other validation statistics based on half-maps FSC may be under- or overestimated. Here, we describe the limitations of the current deposition guidelines and propose an alternative: depositing cryo-EM results in Fourier (reciprocal) space together with the mandatory deposit of molecular masks or their descriptors. This approach addresses the current limitations, preserves critical information from the reconstruction process, and better supports downstream analyses. HighlightsO_LIReciprocal-space deposition to preserve signal and uncertainty estimates. C_LIO_LIMandatory molecular masks deposition for accurate and unbiased FSC validation. C_LIO_LILimitations of half-map-based Fourier Shell Coefficients. C_LIO_LIThe difference maps can be calculated. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/690836v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1a35884org.highwire.dtl.DTLVardef@1086af4org.highwire.dtl.DTLVardef@1a5f118org.highwire.dtl.DTLVardef@177815a_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Otwinowski, Z., Bromberg, R., Guo, Y., Borek, D.. 2025-11-28. Preserving Information and Integrity in Cryo-EM: A Fourier-Space Deposition Approach. https://doi.org/10.1101/2025.11.26.690836

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