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. 2013 Dec;135:24–35. doi: 10.1016/j.ultramic.2013.06.004

Fig. 3.

Fig. 3

Results of processing of 43758 single particle images of beta-galactosidase recorded at 300 keV on the FEI Falcon II detector, using four different procedures. The FSC from the particle data set (red symbols) is compared in each case with that obtained from the same data set with HR-noise substituted beyond 10 Å (blue symbols) or in one case beyond 8.5 Å. Overfitting is shaded blue, with the difference between the two curves, representing correlations between real features of the structure, shaded pink. (a) Results of Frealign processing. To produce more noticeable overfitting, a value of −200 Å2 for the parameter RB-factor was used, noting that this is explicitly not recommended for normal practice [9]. Data out to 7 Å resolution was used in orientation determination, so the overfitting is only evident between 10 and 7 Å resolution. Calculation of FSCtrue from FSCt and FSCn demonstrates a resolution around 6.9 Å. In this case, the very small degree of overfitting does not affect the estimated resolution. (b) The Relion package has been used with complete separation of the data into two halves and gold-standard FSC weighting to carry out low-pass filtering of the reference at each cycle as described [44]. With the gold-standard procedure and gold-standard FSC weighting, there is no overfitting, confirmed by values of FSCn that are zero beyond 10 Å. The map shows 6.4 Å resolution. (c) The Xmipp package has been used with a single reference and “sub-optimal FSC” weighting to apply low-pass filtering at each cycle. With “sub-optimal” FSC weighting however, some overfitting and exaggerated resolution is seen. (d) Results of processing the same data using a new program still under development (McMullan, unpublished) and configured to show substantial overfitting when refined out to 5 Å. FSCtrue (green symbols) shows the true resolution of the structure after removing the effect of overfitting on FSCt. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)