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  1. Evaluation Summary:

    This manuscript describes a tour-de-force approach to determine how the five subunits of the human tRNA ligase complex interact with each other. The authors combine X-ray crystallography of individual protein domains with combinatorial reconstitution analysis and cross-linking mass spectrometry to define and purify a minimal tRNA ligase complex core. Their reductionist and highly analytical approach produces a treasure-trove of data with exceptional quality, the true value of which will become apparent once additional structural information (from e.g. cryo-electron microscopy) becomes available, enabling independent biochemical validation.

    (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 #2 and Reviewer #3 agreed to share their name with the authors.)

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  2. Reviewer #1 (Public Review):

    The authors present a technically accomplished multi-modal study of the human tRNA ligase complex entailing inter- and intra-subunit contact mapping by crosslinking/mass spec, protein truncations to delineate sufficiency for protein-protein interactions, a crystal structure of the N-domain of the CGI-99 subunit, delineation of a minimal active ligase complex, and a crystal structure of the RTCB ligase subunit.

    The work is clearly of general interest. It provides a foundation for future efforts to solve the structure of the entire tRNA ligase complex.

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  3. Reviewer #2 (Public Review):

    Kroupova and colleagues present the manuscript "Molecular Architecture of human tRNA ligase complex" which describes the first detailed dissection of the structure and assembly of the essential, multi-subunit tRNA ligase complex. The authors present, alongside crystal structures of the Rtcb catalytic subunit and the N-terminus of the associated CGI-99 subunit, a comprehensive deletion analysis and mass spectrometry investigation of the composition and assembly of the entire hetero-oligomeric assembly, identifying a novel sub-assembly between the CGI-99 and FAM98B subunits. The study is elegant, beautifully presented and written, easily followed and interesting, providing a high-quality and important dissection of this essential complex.

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  4. Reviewer #3 (Public Review):

    The tRNA ligase complex participates in protein (endonuclease & ligase)-mediated RNA splicing, which contrasts with the better known RNA-mediated splicing in the context of the spliceosome. Protein-mediated RNA splicing may date back to the ancient RNA-protein world where it might have served to defend RNA against invading introns. RTCB-like RNA ligases such as found in the human tRNA ligase complex act in numerous cellular pathways including bacterial RNA repair, and they follow a highly interesting and complex but structurally poorly understood mechanism that joins RNA 5'-hydroxyl ends with 3'-ends that carry a 2'-3' cyclic phosphate. Although the components of the human tRNA ligase complex are known and although there is crystal structural information on archaeal RTCB homologs, the function of the non-enzymatic components and the structural organization of the complex are unknown.

    The quality of the data in Kroupova et al. and their presentation in the manuscript is outstanding, which is easy to follow even for general readers. The authors carefully avoid to over-interpret their cross-linking data, although some remarks on this as outlined in my detailed remarks may benefit readers who are less familiar with this method. The methods are described with exceptional detail and information and will be extremely useful to scientists who plan similar approaches for their own protein complexes of choice and especially if these are still too undefined and/or flexible to be amenable for direct structural analysis by X-ray crystallography or single-particle cryo-electron microscopy.

    This is a prime example for an analytic biochemical approach with modern methodology to a challenging problem in structural biology.

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