RR:C19 Evidence Scale rating by reviewer:
Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.
SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Three putative ion channels (E, 8a, and 3a) are encoded by the virus and be involved in viral assembly and release. Understanding these ion channels’ structure and function can advance our knowledge of the virus, and facilitate the development of new therapies against SARS-CoV-2.
In this manuscript, Kern et al. reported cryo-EM structures of a dimeric and a tetrameric SARS-CoV-2 3a in lipid nanodiscs at 2.1Å and 6.5Å resolution, respectively. They found that 3a adopts a novel fold, which has never been reported before, with a large polar cavity across half of the membrane. There are separate water- and lipid-filled openings that lead to the cytoplasm and surrounding bilayers. Electrophysiology and fluorescent ion imaging studies revealed that 3a is a Ca2+-permeable non-selective cation channel. Point mutations of the polar cavity revealed the location for ion permeability and the authors have also screened for polycationic inhibitors of the 3a channel activity.
The manuscript is well written and easy to understand. The dimeric and tetrameric structures have revealed how the SARS-CoV-2 3a protein is folded and interacts with lipids. It is exciting that they have successfully implemented Sjors H.W.Scheres method using a new electron gun, energy filter, and Facon 4 direct electron camera to reach a resolution near 2Å even with a membrane protein of ~60kDa, such as 3a. Nevertheless, the following comments and questions should be addressed to further improve the manuscript:
1. The nanodisc sample and proteoliposomes were reconstituted using different types of lipids. The authors should check whether there are any structural differences if similar lipids are used. In this manuscript, DOPE and MSP1E3D1 were found to be intact with 3a. Since lipids often have a significant impact on structures or conformation of membrane protein, it would be useful to test a different lipid composition.
2. The authors claimed that ~10-30% of the protein is a tetramer, which is a biochemically stable species, independent of protein concentration. It is unclear which form, the dimeric or the tetrameric plays the role of the real ion channel. Is a dimer alone sufficient to function as the ion channel? If so, what is the physiological role of a tetramer? Is it necessary for ion uptaking?
3. For the emodin sample, it’s better to compare differences between the continuous TM map (derived from ~25% particles) and discontinuous map( from ~75% particles), especially in the transmembrane region. Does this discontinuous map also exist in the apo 3a sample? If not, is it possible that emodin may have some impact on the transmembrane domain?
4. Although the structures have revealed a polar pocket across half the membrane, it is still not entirely clear how the 3a protein functions. Since the authors have already identified polycations as 3a channel inhibitors, it may be worth comparing any structural differences between the apo 3a sample and the 3a-polycations complex sample.