This manuscript by Poweleit et al. Here the authors isolate the native ESX-3 complex from M. This is a beautiful and informative structure, and the analysis is rigorous. The work is significant because it corroborates the structural observations recently published by a competing group Famelis et al. Thank you for submitting your article "The native structure of an ESX translocon" for consideration by eLife.
Your article has been reviewed by four peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Richard Aldrich as the Senior Editor.
The reviewers have opted to remain anonymous. The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. A virtually identical structure of the same complex has been published in Nature by Famelis et al. In contrast to the structure published by Famelis et al. It was felt that the eLife policy on "scoops" protects the authors of this manuscript and that it therefore should be considered further after suitable revisions.
These publications have reported higher order oligomers in multiple mycobacterial species M. Importantly, Beckham et al. In the current study it is unfortunately not mentioned which SEC fractions were used for the cryo-EM analysis, but it is suspected that the void peak fractions were not analyzed. To confirm that the native ESX-3 complex forms higher order oligomers, the authors should more thoroughly investigate the presence of these multimers, e.
Indeed, the presence of such a large cavity in between these two subunits is striking. However, the surface of this cavity consists, except for a number of polar residues, solely of hydrophobic residues. In agreement with a hydrophobic interface, the structure reveals extra densities at the periplasmic part of the cavity suggesting the presence of lipids or detergent. The cytoplasmic half of the cavity does not reveal these extra densities, which could be caused by the presence of several polar residues here, as also discussed by the authors.
However, lipids in this part of the cavity could have been removed during solubilization of the complex. To show that partially lipid-filled cavities have been observed also in other membrane complexes, the authors refer to a publication describing the presence of a similar lipid plug in the central cavity of a reconstituted rotor cylinder of the ATP synthase Meiers et al.
Febs Lett However, it is explicitly mentioned in this article that "As the detergent-purified c cylinder is completely devoid of phospholipids, these are incorporated into the central hole from one side of the cylinder during the reconstitution procedure". It therefore remains unclear whether such lipid-plugged membrane pores actually exists in vivo.
In addition, Poweleit et al. It is difficult to envision though how lipids would be able to mediate translocation of folded substrate dimers with mostly hydrophilic surfaces. This would be a completely new mechanism of protein translocation not seen for any other protein secretion machineries.
In addition, the authors speculate even further that besides hydrophilic proteins also hydrophobic molecules, such as lipids, could be transported through this cavity, while there is no clear evidence that type VII secretion systems are able mediate lipid transport.
In conclusion, while it is indeed striking that such a large cavity is present in the ESX-3 complex, the authors should more cautiously discuss this model, by also mentioning the major problems with this model as discussed above. The translocation model of substrates through the central pore of a hexameric complex is far less speculative, as it is supported by experimental data, published by several groups as also mentioned in the Discussion.
This model should therefore get more emphasis. The requirement for this extraordinarily large movement of the ATPase domains makes it difficult to presuppose the path of the ATPase domains during the secretion cycle.
In our revision we comment on this issue in the Discussion and alter the model figure revised Figure 6 to reflect the need for these molecular motions. These micrographs contain a majority of aggregated particles and are challenging to analyze, which is why we left them out of the first submission. However, there is a small population of monodispersed particles, which we have analyzed. In terms of the size, these particles could be interpreted as a hexameric form, but given the excellent distribution of views we observe in the dimer form we were surprised not to observe any evidence in the 2D class averages that would suggest symmetry higher than 2.
It is certainly possible that these particles do not represent a fixed state but rather misincorporation of several dimers into a single micelle. We present these data in a new supplemental figure Figure 1—figure supplement 4. We have also attempted to crosslink the purified protein before running on the SEC and we do not see evidence of additional, significant crosslinking between dimers, in the purified preparation.
We include this gel in Author response image 1 but not in the resubmitted manuscript. As the reviewers note, the presence of a large cavity in the inner membrane, formed by the EccD dimer, is very unusual. In searching the literature and doing extensive homology searches, we have discovered no clear examples of similar membrane protein structures.
Although we do believe that this cavity will eventually be shown to be of functional importance, as the reviewers appear to recognize, to fully understand the native state and function of EccD is beyond the scope of the current work. We have thus rewritten the Results and Discussion section to be more descriptive and less speculative. We have removed the reference to the c cylinder as we do not want to confuse the reader with a loose analogy that may not provide useful evidence.
We have restructured and rewritten the Discussion to further emphasize the relationship between the prior literature and the oligomer model.
Our intension with this language was to point out the interactions between the cytoplasmic domain of EccD 3 and the other two proteins with significant cytoplasmic domains, EccE 3 and EccC 3. EccD 3 contains a highly conserved region in the linker connecting the ubiquitin-like domain at the N-terminus to the transmembrane region. Despite the strong conservation, the interface made by this region each copy of EccD 3 in the homodimer is strikingly different.
In one case, the region interacts extensively with the other cytoplasmic domain of EccD 3 as well as with EccE, anchoring EccE in a rigid conformation. In contrast, this region has a completely different conformation in the symmetry related copy of EccD, where it reaches in a different direction to form a nexus of interactions with EccB, and EccC.
Given the need for the rearrangement of EccC to accommodate the R-finger mechanism of the ATPase 1 domain, we speculate that flexibility of the EccD 3 linker could allow for the release of EccE and EccC from their rigid positions, thus allowing for a rearrangement of the ATPase domains into an active conformation. To respond to the comments. National Center for Biotechnology Information , U.
Journal List eLife v. Published online Dec Author information Article notes Copyright and License information Disclaimer. Oren S Rosenberg: ude. Received Oct 23; Accepted Dec This article is distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use and redistribution provided that the original author and source are credited.
This article has been cited by other articles in PMC. Supplementary file 2: Top Dali server hits. Supplementary file 3: Buried surface area. Transparent reporting form. Abstract The ESX or Type VII secretion systems are protein export systems in mycobacteria and many Gram-positive bacteria that mediate a broad range of functions including virulence, conjugation, and metabolic regulation.
Research organism: Other. Results A major innovation made possible by the dramatic improvements in cryo-EM Cheng, is the ability to examine challenging protein samples at atomic resolution, even when samples are only available at low concentrations. Open in a separate window. Figure 1. Overview of the ESX-3 tagging, purification, and structure. Figure 1—figure supplement 1. Figure 1—figure supplement 2.
ESX-3 dimer purification optimization. Figure 1—figure supplement 3. Examination of the void volume. Figure 1—figure supplement 4. Initial data collection and initial model generation. Figure 1—figure supplement 5. Data processing workflow for final data collection. Figure 1—figure supplement 6. Consensus and focused refinements. Table 1. Data collection and refinement statistics. Figure 2. The structure of EccD 3. Figure 2—figure supplement 1.
EccD 3 map and model. EccC 3 and EccE 3 make extensive, stabilizing interactions with the asymmetric, cytoplasmic domains of EccD 3 The next component of the stable upper cytoplasmic region is EccE 3.
Figure 3. The structure and protein-protein interactions of EccE 3. Figure 3—figure supplement 1. EccE 3 map and model. Figure 4. The structure and protein-protein interactions of EccC 3. Figure 4—figure supplement 1. EccC 3 map and model.
Figure 4—figure supplement 2. Conformational differences between protomer i and protomer ii. EccB 3 extends into the periplasm and stabilizes dimer formation The ESX-3 dimer is stabilized by cross-protomer interactions formed by the two EccB 3 proteins. Figure 5. The periplasmic multimerization domain. Figure 5—figure supplement 1. EccB 3 maps and models. Discussion The ESX-3 structure presented here is purified without the addition of substrates or nucleotide.
Figure 6. Two models of the ESX-3 translocon complex. Figure 6—figure supplement 1. A hexameric model of the ESX-3 dimer. Protein purification Purification for high resolution structural determination: M. Atomic model building The cytoplasmic domain from the crystal structure of EccD 1 PDB 4KV2 was docked into the cytoplasmic domains of the two EccD 3 molecules and the sequence was mutated.
Low resolution modeling The left and right protomer map, periplasmic focused refined map, and lower cytoplasmic focused refined map were all docked into the consensus map and added together using chimera. Funding Statement The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Additional information Competing interests No competing interests declared. Additional files Supplementary file 1.
Model Refinement Statistics. Click here to view. Supplementary file 2. Top Dali server hits. Supplementary file 3. Buried surface area. Transparent reporting form Click here to view. Acta Crystallographica.
Section D, Structural Biology. UCSF pyem. ConSurf an improved methodology to estimate and visualize evolutionary conservation in macromolecules. Nucleic Acids Research. Systematic, multiparametric analysis of Mycobacterium tuberculosis intracellular infection offers insight into coordinated virulence.
PLOS Pathogens. Nature Microbiology. Systematic genetic nomenclature for type VII secretion systems. Esx systems and the mycobacterial cell envelope: what's the connection? Journal of Bacteriology. Current Pharmaceutical Design. Current Topics in Microbiology and Immunology. Error when resizing a datastore: Invalid Size, Size Must be a multiple of This was a regression and has been fixed.
Snapshot info missing when creating VMFS datastore from snapshot. Old plugin showed the snapshot information during the wizard, this has been added back.
Deletion of datastore requires manual rescan when host in maintenance mode. Maintenance mode hosts were skipped when removing datastores, they are now cleaned up. Unable to create datastore in maintenance mode. If the chosen host was in maintenance mode, the creation would fail. This is now supported. Known issues Plugin does not offer custom per-feature permissions. First release also certified with vSphere 6. Fixed issues Errors in datastore provisioning wizard around loading FlashArray list, matching host groups, or creating the datastore, such as " String index out of range:" This was due to introduced code for NVMe-oF based datastores that caused lookups to fail if the type was unknown.
This is fixed. Any customer experiencing generic "could not lookup object errors" should upgrade to this release. Multiple pathologies relate to this issue. The namespace ID is now correctly shown. Fixed issues Changed passwords could appear in debug logs of vSphere. Leading or trailing white space automatically removed when pasting a JWT into the Pure1 authentication Invalid Pure1 key will show true error message from Pure1 Improved vVol datastore mounting workflow Datastore size allowed non-numeric numbers Fixed bad error messages with vVol datastore mounting.
Certified only for vSphere 6. Specific errors will be properly surfaced up to the user if authentication fails. Patch releases ex. Customers looking for certified releases only should wait for the next minor release ex. Remove line breaks manually if they exist through a text editor like Notepad. This situation will be automatically handled in a future release. The workaround is to upgrade to 4. Improved Error Reporting when Creating a VVols Datastore on a Cluster When attempting to create a VVols Datastore on a Cluster whose members are only partially configured on the FlashArray, an error message is displayed indicating which hosts need to be configured for the operation to complete.
Previous releases of the plugin would attempt to configure the VVols storage provider on all vCenter instances in an enhanced linked-mode configuration. Create datastore volumes in volume groups This release of the plugin includes support for creating datastores with the backing volume in a volume group. The user can now provide the volume name including the volume group when creating the datastore and the datastore backing volume will be automatically added to the volume group in Purity.
This version of the plugin fixes this issue. Snapshot revert to datastore of a different size Past versions of the plugin would not check or warn when a user attempted to revert a datastore to a snapshot of a different size than the current datastore. This version of the plugin checks and prevents these operations.
Known Issues RBAC with group permissions does not work If a group is granted RBAC permissions, users within the user group may not be able to perform the actions to which the group has privileges granted. As a workaround, users should be given direct permissions when using RBAC rather than through a group.
Concurrent plugin operations may fail on linked-mode vCenter servers All past and present versions of the plugin may exhibit failures when used simultaneously by 2 or more vSphere web client users on vSphere servers configured in linked-mode. Failures may report unrelated error messages and may require a retry.
We reiterate that these failures only manifest when the plugin is used to manage vSphere servers in linked-mode and will not affect any standalone vSphere configuration. Plugin installation may fail due to invalid TLS options set by third-party plugins Plugin installation may fail because of the global TLS options configured by other third-party vSphere Web Client plugins installed on your vCenter server.
If the global TLS options have been set to version 1. Creating a VMFS Datastore from a snapshot does not always allow connection to same hosts When creating a new VMFS datastore from a copy of a FlashArray snapshot in the datastore view, the dialog may not allow connecting the new datastore to all hosts which are connected to the original datastore the snapshot originates from if the original datastore is mounted on a subset of a host cluster. As a workaround, the datastore may created on a single selected host and then manually connected and mounted on additional hosts after creation.
In a future version of the plugin, we intend to provide a full list of valid hosts to optionally connect the newly created datastore to. A single vCenter instance which is not online or accessible may cause some configuration operations to fail, including adding a new FlashArray to the plugin.
To clear this message, load the web UI of the FlashArray directly in a browser window, accepting the certificate. Refresh the plugin browser window and the FlashArray UI should now display. These users are invalid choices for RBAC and should be ignored. Registering Storage Providers with vCenter 6. This does not affect vCenter 6. What's New vSphere 6.
Users requiring support for vSphere 6. Graham July 20, July 21, NSX VMware. Graham June 26, August 27, This website uses cookies to improve your experience. Accept Read more Privacy Policy. Close Privacy Overview This website uses cookies to improve your experience while you navigate through the website. Out of these, the cookies that are categorized as necessary are stored on your browser as they are essential for the working of basic functionalities of the website.
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