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One caveat inherent to the thresholding method is that simple operations such as filtration and masking can alter the values of voxels inside or outside the missing wedge in different ways, leading to misidentification of missing wedge voxels and therefore increasing subtomogram alignment error.
The potential artifacts that are caused by the missing wedge in the EM data could have affected this particular conclusion, especially the connections between the filaments, as the authors also showed in their simulations.
For single-axis tomograms, the accuracy of tracing is lower (16% false negatives and 14% false positives) due to the missing wedge in electron tomography.
Data collection methods alternative to canonical single-axis ET have been proposed to reduce the deleterious effects of the missing wedge in cryoET, such as dual-axis tomography (Penczek et al. 1995; Mastronarde 1997; Tong et al. 2006; Xu et al. 2007), conical tilt (Lanzavecchia et al. 2005), and multiple-axis tomography (Messaoudi et al. 2006).
Consequently, there is a "missing wedge" in the data.
The long-standing problem of the missing wedge in electron tomography caused by our current inability to tilt the specimen much beyond 65° makes it impossible to follow features all the way around the cell's perimeter.
Similar(54)
Hence, the finding of complete rings is supported by the fact that perfect coincidence of any hypothetical gaps in constricting rings with the missing wedges in all of the analysed tomograms, found after all in entirely random orientations, would be remarkably implausible.
Although we cannot exclude that the density corresponding to Gic1 appears more spread out due to missing wedge artifacts in our tomograms, the average large volume of the Gic1 density in the electron tomograms indicates that it must contain multiple Gic1 molecules.
Additionally we included some sentences that explain the limits of cryo-ET, such as missing wedge artifacts, in the revised manuscript.
Since the missing wedge artifact in this geometry causes only the elongation of the septin filaments, their exact tracing was straightforward, but the cables were often too short.
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.
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