Structure of an oxygen-induced tubular nanocompartment in Pyrococcus furiosus
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eLife Assessment
Using advanced CryoEM and mass spectrometry, the authors provide compelling evidence of how tubule formation occurs in an oxygen-dependent manner. These fundamental findings offer a novel mechanism by which rubrerythrin tubules encapsulate encapsulin to prevent oxidative stress in Pyrococcus furiosus. However, there are a few reasonable concerns about biochemical validations and the lack of adequate description of results and methodology.
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Abstract
Abstract
Reactive oxygen species (ROS) pose a significant threat to biological molecules, prompting organisms to develop systems that buffer oxidative stress and contain iron, which otherwise amplifies ROS production. Understanding oxidative stress responses requires identifying the key proteins involved and their cellular organization. Here, we combined proteomics and cryo-EM to investigate the response of the anaerobic hyperthermophilic archaeon Pyrococcus furiosus to oxygen exposure. Proteome analysis revealed a significant upregulation of the oxidoreductase Rubrerythrin (Rbr) under oxidative stress. Cryo-electron tomograms showed the formation of prominent oxidative stress-induced tubules (OSITs). Single-particle cryo-EM and mass spectrometry of enriched OSITs identified them as stacked rings of Rbr homotetramers. The 3.3 Å structure demonstrates that rubredoxin-like domains mediate homotetramer assembly, suggesting that their oxidation drives OSIT formation. Within OSITs, we discovered virus-like particles formed by a ferritin-like/encapsulin fusion protein with iron hydroxide cores, uncovering a sophisticated organelle that protects P. furiosus from ROS through advanced compartmentalization.
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eLife Assessment
Using advanced CryoEM and mass spectrometry, the authors provide compelling evidence of how tubule formation occurs in an oxygen-dependent manner. These fundamental findings offer a novel mechanism by which rubrerythrin tubules encapsulate encapsulin to prevent oxidative stress in Pyrococcus furiosus. However, there are a few reasonable concerns about biochemical validations and the lack of adequate description of results and methodology.
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Reviewer #1 (Public review):
Summary:
It is now increasingly becoming clear that macromolecules and their complexes can form larger structures such as filaments or cages in the cells under certain conditions. These can be beneficial for the cells to promote and coordinate metabolic activity or result in protection against stress. Reactive oxygen species (ROS) can be damaging to macromolecules in cells that grow both aerobically and anaerobically, and they have evolved different mechanisms to cope with ROS. Aerobic organisms have a number of enzymes to combat ROS, while anaerobic organisms have evolved other means, and one such mechanism is described by Song et al in the article.
In Pyrococcus furiosus, a hyperthermophilic anaerobic bacterium, Song et al describe the formation of Oxidative stress-induced tubular structures (OSITs). Using …Reviewer #1 (Public review):
Summary:
It is now increasingly becoming clear that macromolecules and their complexes can form larger structures such as filaments or cages in the cells under certain conditions. These can be beneficial for the cells to promote and coordinate metabolic activity or result in protection against stress. Reactive oxygen species (ROS) can be damaging to macromolecules in cells that grow both aerobically and anaerobically, and they have evolved different mechanisms to cope with ROS. Aerobic organisms have a number of enzymes to combat ROS, while anaerobic organisms have evolved other means, and one such mechanism is described by Song et al in the article.
In Pyrococcus furiosus, a hyperthermophilic anaerobic bacterium, Song et al describe the formation of Oxidative stress-induced tubular structures (OSITs). Using proteomics and electron cryomicroscopy (CryoEM), the authors find that the protein Rubrerythrin is upregulated upon exposure to oxygen, and the tetramer of this protein assembles to form these tubules that are varied in length with a consistent diameter of ~480 Å. They further observe that some of these tubules also have spherical viral-like particles. With enriched fraction of the OSITs from the cells and proteomics, it is shown that the predominant protein is encapsulin, which forms a caged structure and traps ferric iron. The combined structures of OSIT by rubreerythrin and the VLPs of encapsulin protect the cells from oxygen radicals by forming a complex.Strengths:
The combination of proteomics and electron microscopy with the employment of both tomography of cellular sections and single particle cryoEM of enriched samples.
Weaknesses:
Some description of the methods, in particular the workflow of image processing, is not easy to follow and can be described with more clarity and be easier for non-experts to read/understand.
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Reviewer #2 (Public review):
The manuscript entitled "Structure of an oxygen-induced tubular nanocompartment in Pyrococcus furiosus" by Wenfei Song et al. employs whole-cell mass spectrometry and cryo-EM (including tomography, helical reconstruction, and single-particle analysis) to investigate the structure and function of the oxidoreductase Rubrerythrin (Rbr) from Pyrococcus furiosus. The study reports that under oxidative stress, Rbr forms a tubular structure, in contrast to its behaviour under anaerobic conditions. Authors characterized oxidoreductase Rubrerythrin (Rbr) from Pyrococcus furiosus under anaerobic conditions and formed a tubular structure when induced with oxidative stress. This study is well-designed. However, I have several questions related to the experimental design and the results obtained from those experiments, …
Reviewer #2 (Public review):
The manuscript entitled "Structure of an oxygen-induced tubular nanocompartment in Pyrococcus furiosus" by Wenfei Song et al. employs whole-cell mass spectrometry and cryo-EM (including tomography, helical reconstruction, and single-particle analysis) to investigate the structure and function of the oxidoreductase Rubrerythrin (Rbr) from Pyrococcus furiosus. The study reports that under oxidative stress, Rbr forms a tubular structure, in contrast to its behaviour under anaerobic conditions. Authors characterized oxidoreductase Rubrerythrin (Rbr) from Pyrococcus furiosus under anaerobic conditions and formed a tubular structure when induced with oxidative stress. This study is well-designed. However, I have several questions related to the experimental design and the results obtained from those experiments, which are listed below.
(1) The authors have mentioned that "Under aerobic conditions, Rbr levels are 3 to 13 times higher compared to anaerobic conditions (Figures 1a-d)." Also, they performed whole-cell mass spec to measure the overexpression of the Rbr enzyme under anaerobic conditions. Thus, from the above statement, I consider the authors' claim that P. furiosus cells were cultured under anaerobic conditions and then exposed to oxidative stress. While cell growth under anaerobic conditions appears perfectly fine, the authors conducted the rest of the experiment under aerobic conditions during mass spectrometry and cryo-EM sample preparation. As a baseline, the author first grew the cells in their preferred anaerobic environment and also imaged the same cells that were exposed to air (aerobic) after anaerobic growth. The cell growth in anaerobic conditions is perfectly fine. But how did authors make sure that during anaerobic conditions, the Rbr enzyme is not expressed or not formed? As a control experiment, authors should demonstrate that during mass spec and cryo-EM sample preparations, cells are not exposed to air or maintained in an anaerobic environment. From anaerobic conditions, whenever cells were selected for spec and cryo-EM, cells were exposed to O2, and definitely controlled cells were not in anaerobic conditions anymore.
The authors collected P. furiosus wild-type or Rbr knockout cells in an anaerobic hood, but after that, they centrifuged the cells and plunged them using a Vitrobot. Are the instrument, centrifuge, and Vitrobot kept in an anaerobic environment? Recently, a few studies (anaerobic plunge-freezing in cryo-electron microscopy, Cook et al. (2024), Hands-Portman and Bakker (2022) DOI: 10.1039/D2FD00060A ) have mentioned the anaerobic plunge freeze setup for protein sample or cell freezing. I guess the authors did not use that setup. In these circumstances, the cell is already exposed to O2 during centrifugation and Vitrobot freezing. How were the control experiments properly performed in anaerobic conditions? A similar argument is true for Lamella grid preparation, where the enzyme was already exposed to O2, and single-particle grid preparation, where the purified enzyme is already exposed to O2. How were the control experiments properly performed in anaerobic conditions?
(2) It is important to provide evidence that the overexpressed protein is actually in an anaerobic condition and is later induced with more O2. Also, authors should confirm biochemically that the overexpressed protein in their desired protein "oxidoreductase Rubrerythrin (Rbr)". No biochemical data were provided in this manuscript. During single-particle analysis, the authors had to purify the protein sample and confirm that these were their desired protein samples. No biochemical or biophysical experiments were performed to confirm that the overexpressed protein is the desired protein.
(3) Figure 3, the atomic model looks different in all four tetramers. However, I have fitted the atomic model into the cryo-EM map, which looks reasonable. However, it will be easier for the reader to evaluate the model if the authors show different orientations of the atomic model, as well as if the authors could show that the atomic model fits the cryo-EM map.
(4) How did the authors select initial particle sets like 24 lakhs when forming helices and not forming isolated particles?
(5) The authors proposed a model for electron transfer upon oxidative stress. However, the data is not convincing that VLP is surrounded by Rbr and forms a tube-like structure. Generally, VLP is a sphere-like structure, and Rbr can form a tube-like structure when it interacts with spherical VLP. Rbr will surround VLP, and it will form a Rbr-decorated sphere-like structure.
(6) It will also be important to comment on the diameter of Oxidative stress-induced tubules (OSITs) and 3D reconstruction and/or helical reconstruction of purified protein samples. The spherical cyan densities within the tube are not very clear. If VLP is surrounded by Rbr (Figure 4), extra Rbr densities will be observed on VLP in the tomogram (in Figure 1). However, in the tomogram, VLP is inside Oxidative stress-induced tubules (OSITs). Figure 1 is a contradicting Figure 4. The authors should explain it properly.
(7) The authors performed helical reconstruction. Where is the Layer line calculation in helical reconstruction, and how do authors identify helical parameters for reconstruction?
(8) The authors used an extremely confusing methodology, which was very difficult to follow. The authors performed tomography, helical reconstruction, and single-particle analysis. Why did the authors need 3 different image processing methods to resolve structures that are not clear to me? The authors should also show the proper fitting between the map and the model. In Supplemental Figure 6c, the overall fitting of the subdomain looks ok. However, many peptide chains and side chains are not fitted properly in the EM density map. It will be helpful to show proper side chain fitting. In Supplementary Fig. 6a, the authors binned the data (Bin 8 or Bin 2) but did not mention when they unbinned the data for data processing. Also, the authors implemented C2 symmetry during local refinement. Why do authors suddenly use C2 symmetry expansion?
Minor Comments:
(1) The authors should properly show a schematic diagram of the enzyme subdomains. It will help to understand interactions or tetrameric assembly.
(2) The introduction is poorly written. It will really be helpful for the reader if the authors provide a proper introduction.
(3) The atomic model did not fit into the cryo-EM, so it was hard to determine the overall fitting.
(4) 17.1A pixel size? It's surprising.
(5) It will be better to calculate local resolution and show the map's angular distribution. It is obvious that resolution at the peripheral region will be poorer than core region. Therefore, it will be better to calculate local resolution. Additionally, authors should show the map to model fitting.
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Reviewer #3 (Public review):
Summary:
The manuscript authored by Song et al explores the oxidative stress response of Rubrerythrin in Pyrococcus furiosus and the formation of unique tubules that also encapsulate Encapsulin VLPs. This is an excellent study employing diverse methods to comprehensively study the formation of these assemblies under oxidative stress and lays the foundation of understanding oxidative stress through the formation of tubules among redox-sensing proteins like Rubrerythrin. The authors decipher the molecular structure of the tubules and also present a high-resolution reconstruction of the rubrerythin unit that forms the OSITs.
Strengths:
The study is done thoroughly by employing methods like cryoET, single particle cryoEM, mass spectrometry, and expression analyses of knockout strains to delve into an important …
Reviewer #3 (Public review):
Summary:
The manuscript authored by Song et al explores the oxidative stress response of Rubrerythrin in Pyrococcus furiosus and the formation of unique tubules that also encapsulate Encapsulin VLPs. This is an excellent study employing diverse methods to comprehensively study the formation of these assemblies under oxidative stress and lays the foundation of understanding oxidative stress through the formation of tubules among redox-sensing proteins like Rubrerythrin. The authors decipher the molecular structure of the tubules and also present a high-resolution reconstruction of the rubrerythin unit that forms the OSITs.
Strengths:
The study is done thoroughly by employing methods like cryoET, single particle cryoEM, mass spectrometry, and expression analyses of knockout strains to delve into an important mechanism to counter oxidative stress. The authors perform comprehensive analyses, and this study represents a vital contribution to understanding how anaerobic organisms can respond to oxidative stress.
Weaknesses:
Not all encapsulin particles seem to be inside the OSITs. Do the authors have any insights into how the tubules sequester these viral particles? Do the VLPs have a role in nucleating the OSIT assembly, and are there interactions between VLP and OSIT surfaces? These could be points that can be discussed in greater detail by the authors.
Can the authors get a subtomogram averaging done for the encapsulin VLPs? A higher resolution reconstruction may provide potential interaction details with the OSITs, if there are any.
The role of the dense granules observed in the rubrerythrin deletion strain is not very well discussed. Is there a way these granules counter oxidative stress? The EDX scanning seems to show a Phosphate increase similar to Ca and Mg. Are these aggregates therefore likely to be calcium and Mg phosphate aggregates? This section of the paper seems incompletely analysed.
The authors should provide density and coordination distances around the diiron ions and provide a comparison with available crystal structures and highlight differences, if any, in Figure 3. Local resolution for the high-res map may be provided for Supplementary Figure 6.
Overall, this is a well-performed study with clear conclusions. The discussion points need to be improved further.
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