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Non-Uniformity of Projection Distributions Attenuates Resolution in Cryo-EM

By Philip R. Baldwin, Dmitry Lyumkis

Posted 13 May 2019
bioRxiv DOI: 10.1101/635938 (published DOI: 10.1016/j.pbiomolbio.2019.09.002)

Virtually every single-particle cryo-EM experiment currently suffers from specimen adherence to the air-water interface, leading to a non-uniform distribution in the set of projection views. Whereas it is well accepted that uniform projection distributions can lead to high-resolution reconstructions, non-uniform (anisotropic) distributions can negatively affect map quality, elongate structural features, and in some cases, prohibit interpretation altogether. Although some consequences of non-uniform sampling have been described qualitatively, we know little about how sampling quantitatively affects resolution in cryo-EM, especially given the numerous different projection schemes that can arise in experimental situations. Here, we show how inhomogeneity in any projection distribution scheme attenuates the global Fourier Shell Correlation (FSC) in relation to the number of particles and a single geometrical parameter, which we term the sampling compensation factor (SCF). The reciprocal of the SCF is defined as the average over Fourier shells of the reciprocal of the per-particle sampling and normalized to unity for uniform distributions. The SCF therefore ranges from one to zero, with values close to the latter implying large regions of poorly sampled or completely missing data in Fourier space. Using two synthetic test cases, influenza hemagglutinin and human apoferritin, we demonstrate how any amount of sampling inhomogeneity always attenuates the FSC compared to a uniform distribution. We advocate quantitative evaluation of the SCF criterion to approximate the effect of non-uniform sampling on resolution within experimental single-particle cryo-EM reconstructions.

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