Ambiguities in helical reconstruction

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Dr Egelman analyzes the validity of a cryoEM helical reconstruction of the prion-like filament formed by the CARD domain of the RIG-I adaptor molecule MAVS, as reported by Xu et al. in eLife (Xu et al. eLife, 2014, 3:e01489). The study by Xu et al. was published before a related study was published by Wu et al. in Molecular Cell (Wu et al. Molecular Cell 2014, 55:511-524). The situation merits follow-up because the two reported MAVS structures are entirely different. There is no dispute that the Wu et al. study contained high quality EM data, that their reconstruction was technically correct, and that they successfully generated a near-atomic resolution structure of their MAVS filaments. The central question is whether differences in sample preparation generated distinct MAVS filaments with different physical structures, or whether the differences in the reported structures instead reflect methodological problems in the Xu et al study. Dr Jiang (one of the corresponding authors on the Xu et al. paper) has posted a Comment that accompanies the original eLife manuscript in which he argues that the reported differences in the filament helical symmetries and structures reflect genuine physical differences in the MAVS filaments generated by the two groups. Here, Egelman analyzes the central claims of that Comment and also discusses the validity of the original Xu et al. reconstruction.

Egelman downloaded part of the original data deposited by Xu et al. in the EMPIAR data base and performed image analyses to test the arguments put forth by Jiang that the filaments used in the two studies were indeed different. He shows convincingly that the three central pieces of evidence presented in the Jiang Comment are invalid. He makes several important points, including that Xu et al. did not appreciate that their filaments had significant out-of-plane tilt and that convergence stability in a symmetry search is not sufficient to establish the validity of that symmetry. Importantly, he also shows that the Xu et al. structure does not agree with its own PDB model beyond a resolution of ∼20 Å, suggesting that the computed EM density is not correct. These arguments convincingly rebut the points raised in Jiang's Comment, reveal potential inconsistencies in the processing procedure used by Xu et al., and raise substantial doubts about the validity of the Xu et al. reconstruction. These analyses are therefore an important contribution.

The Egelman analysis could be strengthened even further by providing additional evidence that Xu et al. must have imposed the wrong helical symmetry. The author could do this by computing a structure with the symmetry parameters from Wu et al. imposed on the EMPIAR data set from Xu et al. (which he has already been analyzing). Such a structure should contain clearer densities and recognizable secondary structure features.

[…] The Egelman analysis could be strengthened even further by providing additional evidence that Xu et al. must have imposed the wrong helical symmetry. The author could do this by computing a structure with the symmetry parameters from Wu et al. imposed on the EMPIAR data set from Xu et al. (which he has already been analyzing). Such a structure should contain clearer densities and recognizable secondary structure features.

I have been able to improve the resolution that I initially obtained with their images by excluding those with a defocus greater than 3.0μ. This reduced the data set from ∼64k segments to ∼15k segments, but the reconstruction is clearly improved. I have expanded Figure 4 to show a better comparison of the reconstruction from Xu et al. with the correct symmetry with the one from Wu et al. filtered to 12 Å resolution, and shown the atomic model of Wu et al. fit into both volumes.