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  1. Evaluation Summary:

    The manuscript by Yanofsky et al. describes the high-resolution structure of the CIII/CIV super-complex from Mycobacterium smegmatis bound to the anti-tuberculosis drug Q203 which is currently in clinical trials. The authors also provide biochemical data for inhibition of purified CIII/CIV and add important new information regarding the mechanism of Q203. This is an important contribution that will be of broad interest to the field of bioenergetic and tuberculosis, as telacebec represents one of few novel drug classes with potential for treatment of tuberculosis and other mycobacterial infections.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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  2. Reviewer #1 (Public Review):

    A major strength of the paper is the development of an improved activity assay that allows the authors to reliably measure the IC50 of telacebec with their supercomplex preparation.

    A strength of the paper is the development of a 3xFlag-tagged M. smegmatis CIII2CIV2 allowing for one step affinity purification. This purification strategy also allowed the authors to demonstrate that the SOD and LpqE subunits are less tightly associated with the complex and can be lost during size exclusion chromatography. These observations will inform future biochemical work on this complex.

    A strength of the paper is the improved cryoEM reconstruction of the M. smegmatis CIII2CIV2 supercomplex, showing clear density for the LqpE subunit and improved density for the SOD subunit.

    A strength of the paper is the use of 3D variability analysis (3DVA) to identify a subset of particles that have lost one of the LpqE subunits resulting in the Cyt cc subunit to adopt the "open" conformation suggesting a regulatory role for this subunit. Furthermore, the 3DVA reveals movement of the SOD subunit that brings it into proximity of the cyt cc subunit and may allow for direct electron transfer from superoxide to CIV.

    A strength of the paper is the localization of the telacebec biding site to the QP-site of CIII2 and the comparison with the menaquinone bound at the site in the absence of the inhibitor.

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  3. Reviewer #2 (Public Review):

    Previously, these authors reported the structure of the CIII/CIV super-complex from M. smegmatis (Wiseman et al. 2018). In the current manuscript, the cryo-EM structure of CIII/CIV shows different positions for the attached superoxide dismutase and for the characteristic CIII/CIV cc domain, indicating movement of these units during the catalytic cycle.
    The authors confirm that the isolated CIII/CIV is enzymatically active and is inhibited by nano-molar Q203, in line with the drug's MIC and previous experiments using membrane fractions.
    The cryo-EM structure reveals that Q203 binds to the Qp site, which in drug-free CIII/CIV is occupied by a quinone molecule. The structure also shows a network of interactions between Q203 and various residues of CIII/CIV.

    The paper is well written and in general the conclusions are supported by the data.

    To this reviewer, one technical aspect related to the activity assays is unclear: according to the transparent reporting form, six independent assays were performed (technical replicates). Does that mean that the enzyme was purified once and the assay then carried out six times with that purified sample? Usage of multiple biological replicates (i. e. different batches of isolated CIII/CIV) is important, in particular in case, as the authors indicate, some subunits can partially dissociate from the super-complex.

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  4. Reviewer #3 (Public Review):

    Telacebec (Q203) is a novel first-in-class antituberculosis drug that targets Mycobacterium tuberculosis respiration and cellular energy production through inhibition of the mycobacterial cytochrome bcc-aa3 (CIII2CIV2) super complex and thus represents the third class of energetic inhibitors against M. tuberculosis. Q203, currently in phase II clinical trials, has proven anti-tuberculosis activity in humans. Q203 also has the potential to be used in the treatment of nontuberculosis mycobacterial infections e.g. Buruli ulcer, a neglected tropical skin disease.

    The manuscript by Rubinstein and colleagues reports the atomic-resolution entire structure of the Mycobacterial super CIII2CIV2 complex from Mycobacterium smegmatis inhibited by the anti-TB drug Q203. Other groups have reported the structure of CIII2CIV2 complex at high resolution, but none of this previous work has captured the drug inhibited structure. Yanofsky's new CIII2CIV2 complex captures the binding site of Q203 providing the mechanism (protein-inhibitor contacts) of inhibition of the complex and importantly also sheds light on the mechanism of enzyme catalysis. The structure reveals that Q203 binds with its head group deep within the Qp-binding pocket in a pose similar to UQ thus blocking menaquinol oxidation by the complex in both menaquinol binding modes. Multiple interactions are apparent leading to stabilised inhibitor binding.

    The paper provides further structural data on the role of the SOD subunit in the complex and suggests that SOD may indeed transfer electrons from periplasmic superoxide to CIV thus contributing to the PMF and energy generation. The paper is a comprehensive, well written in an easy to follow style and will have broad interest and readership.

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