Ambiguities in helical reconstruction

Abstract

Helical polymers are found throughout biology and account for a substantial fraction of the protein in a cell. These filaments are very attractive for three-dimensional reconstruction from electron micrographs due to the fact that projections of these filaments show many different views of identical subunits in identical environments. However, ambiguities exist in defining the symmetry of a helical filament when one has limited resolution, and mistakes can be made. Until one reaches a near-atomic level of resolution, there are not necessarily reality-checks that can distinguish between correct and incorrect solutions. A recent paper in eLife (Xu et al., 2014) almost certainly imposed an incorrect helical symmetry and this can be seen using filament images posted by Xu et al. A comparison between the atomic model proposed and the published three-dimensional reconstruction should have suggested that an incorrect solution was found.

Keywords: biophysics; cryo-EM; helical symmetry; human; structural biology; three-dimensional reconstruction.

Figure 1.. The three-dimensional power spectrum of a helical filament and power spectra from projections of the filament.

The three-dimensional power spectrum of a helical filament is shown (A). The power spectrum from a cryo-EM image of a helical filament (where the image is a projection of a three-dimensional object onto a two-dimensional plane) is the central section of the three-dimensional power spectrum, and this would be given by the intersection of the red plane in (B) with the power spectrum. The yellow arrows in (B) show the intersection of this plane with a layer plane that arises from a 1-start helix. The intensities on this central section are shown in (D), which would be the power spectrum from untilted filaments, i.e., those whose filament axis is parallel to the plane of projection. The yellow arrow in (D) shows the layer line from the 1-start helix, with peak intensities on both sides of the meridian. Now consider what happens when the filament axis is tilted away from parallel to the plane of projection. The central section (C) would now intersect the 1-start layer plane at the positions given by the two yellow arrows, and in the two-dimensional power spectrum the two peaks would collapse to a single peak on the meridian (E, yellow arrow). If one did not know that the filament was tilted, such intensity would be mistaken for a meridional layer line with the Bessel order n = 0. DOI: http://dx.doi.org/10.7554/eLife.04969.002

Figure 2.. Out-of-plane tilt cannot be ignored in the images of Xu et al.

A histogram (A) of the out-of-plane tilt seen in the filaments of Xu et al. A reconstruction was generated from these filaments while allowing for out-of-plane tilt, and this reconstruction was used as a reference, generating 90 different azimuthal projections (4° increments) for tilt angles from −20° to +20° (also with 4° increments), generating 990 reference projections. The large peaks at both ends of the distribution arise from truncating the search range to smaller values than actually found in the filament population. Using filament segments in the three central bins (−4°, 0°, 4°) for generating a power spectrum (B) it can be seen that there is no meridional intensity on the layer line indicated by the arrow at ∼1/(18 Å), and the appearance of this layer line is now the same as in Wu et al. The log of the power spectrum is shown to better display the large dynamic range. DOI: http://dx.doi.org/10.7554/eLife.04969.003

Figure 3.. In the IHRSR method, the helical parameters (rotation and rise per subunit) are refined each cycle.

The starting parameters were the symmetry determined by Wu et al., and it can be seen that when using the images of Xu et al. these parameters are perfectly stable. The first seven cycles were run with images decimated from 2.3 Å/px to 4.6 Å/px, ignoring out-of-plane tilt. The subsequent cycles were run with the undecimated images and allowing for out-of-plane tilt. DOI: http://dx.doi.org/10.7554/eLife.04969.004

Figure 4.. A reconstruction has been made from the filaments of Xu et al.

(A and C) by applying the helical symmetry described in Wu et al. For purposes of comparison, the reconstruction of Wu et al. (EMDB-5922) has been filtered to 12 Å resolution (B and D). It can be seen that with similar thresholds, both reconstructions show a hole in the center (A and B, arrows), arising from the helical procession of the subunits about the filament axis. The atomic model (3J6J.PDB) from Wu et al. (2014) is shown fit into both reconstructions. DOI: http://dx.doi.org/10.7554/eLife.04969.005

Figure 5.. Comparisons between a model and the actual map are informative.

The reconstruction (A and C) of Xu et al. (EMDB-5890) is compared with their model 3j6c.PDB (B and D). In all four panels (A–D) the same model (3j6c.PDB) is shown as magenta ribbons, but the transparent surfaces in B and D have been generated by filtering the model density to 9.6 Å, the stated resolution of the reconstruction shown in (A and C). The surfaces in (C and D) are shown at a higher threshold than in (A and B). It can be seen that with these thresholds, there is virtually no correlation between the map (A and C) and the model (B and D). A Fourier Shell Correlation between their model and reconstruction (E) shows that at FSC = 0.5 the resolution is ∼22 Å, which is consistent with the visual comparison of the map and model (A–D). DOI: http://dx.doi.org/10.7554/eLife.04969.006