DOI: http://dx.doi.org/10.7554/eLife.03600.008 The orientation of our crystals prevented the full sampling of reciprocal space leading to systematic incompleteness (missing wedge or missing cone).
The lack of detail in the membrane region is unlikely to be caused by the missing wedge as we have observed a similar phenomenon in sub-tomogram averages of other membrane proteins, which have no missing wedge of information, and in single-particle maps when the resolution does not extend beyond 15 Å.
As a result our data contains a wedge-shaped region in Fourier space for which no data has been measured (missing wedge effects), similar to experimental measurements.
Given, the real space subtomogram that excludes any coefficients inside of any of the two missing wedge regions is defined as, where denotes the real part of a complex function; is the Fourier transform operator; and and are missing wedge masks for f and g, respectively.
The limited specimen tilting range that is typically available in electron tomography gives rise to a region in the Fourier space of the reconstructed object where experimental data are unavailable – the missing wedge.
Our simulated subtomograms therefore contain a wedge-shaped region in reciprocal space for which no structure factors have been measured (i.e. the missing wedge effect), resulting in distortions in the density map as observed in the experimental measurements.
The lack of detail in the membrane region is surely due simply to poor resolution perpendicular to the membrane associated with the (quite substantial) missing wedge.
Despite the recent progress in the field of reconstruction and regularization techniques, the missing wedge artefacts remain untreated in most current reconstruction workflows in structural biology.
Fig. 5 Missing wedge reconstruction, processed with NAD. a A WBP reconstruction of aligned, noiseless projections with a 28 ° missing wedge.
Fig. 10 Missing wedge reconstruction processed with Weka. a The perfectly aligned, noiseless 28 ° missing wedge reconstruction from Fig. 5. b The same reconstruction but processed with Trainable Weka Segmentation.
