1. Response to Reviewer #2 (Public Review):

    We thank the reviewer for their encouraging review of our manuscript.

    Considerations:

    1. The cryoEM data showed two main groups of particles: 5-mer protecting 150 bp and a 7-mer protecting either 90bp or 120bp. A few times in the manuscript (both in the results and discussion section) the authors mention a 30-bp MNase digestion ladder is observed. The Mnase data should be included, as this provides evidence that the structures observed by cryoEM indeed represent physiological structures, especially if strong discrete bands are observed at 90, 120, and 150 bp.

    These comments refer to our previous archaeal histone-based chromatin manuscript (Mattiroli et al., Science, 2017), and we have added citations throughout the text to make it clear to the reader that we are referencing previous work.

    1. The two main classes found by cryoEM give the impression that adding dimers results in altered structures. The 7-mer shows an angled structure, which is interpreted as an open structure. The 5-mer shows a more uniform structure, which is interpreted as a closed structure. The former structure protects the full length of DNA on which HTkA histones were reconstituted, whereas the latter might be an incomplete reconstitution or a partially disassembled structure. It also raises the question if the length of the DNA is a limiting factor. What if HTkA was reconstituted on 170 bp or 307 bp instead? Would this in turn only permit the formation of the 5-mer on the 170 bp construct and two 5-mers on the 307 bp construct? The authors should consider addressing this point because the reconstitution might be constrained by the length of the DNA construct used. Indeed, a related topic might be AT content- what does archaeal DNA look like from the perspective of DNA sequence for chromatin (Jon Widom's group had a ChIPSeq paper on this a few years ago, just after his untimely passing).

    In line with comments made by reviewer 1, we have revised the definition of “open” vs “closed” archaeasomes to the more neutral “Class I” and “Class II” (see response to reviewer 1 for details). The effect of DNA length on the number of base pairs that can be maximally wrapped in a continued “closed” supercoil is indeed of interest, and a subject of future studies. For this reason, we hesitate to include too much speculation regarding DNA length, and rather save this for a more involved, data-driven, discussion for our future manuscript. Future work will also address the role of DNA sequence in archaeal chromatin structure.

    1. In the discussion the authors cite that in one archaeal species the Mg2+ concentration is ~120 mM, more than a magnitude greater than that tested in Figure 5. What happens to reconstituted archaeasomes at higher Mg+? This is relevant because in vivo, archaea are thought to have 10x the concentration of Mg+ (amongst other ions) relative to us humble eukaryotes who would probably die of kidney failure at those ionic concentrations. Indeed, at high ionic conditions, eukaryotic chromatin can be made to precipitate out of solution (for e.g. 10mM Mg+, 3M NaCl). An AUC assay with higher Mg2+ concentrations seems a doable and physiologically relevant addition to the ms that would strengthen it. It is relevant to consider that in vivo structure in these halophilic and thermophilic organisms might be dependent on the concentration of various salts and temperature, it would be nice to read the authors' thoughts on this issue.

    While we did not do a full AUC characterization at the high MgCl2+ (> 10 mM) concentrations, we conducted a quick benchtop experiment (via NanoDrop spectrophotometer, data not shown in manuscript) to identify whether 50 mM or 100 mM MgCl2 would cause Arc207 samples to precipitate from solution. Indeed, Arc207 samples do not aggregate at either of these MgCl2+ concentrations, as no absorbance is lost upon centrifuging spiked samples.

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  2. Response to Reviewer #1 (Public Review):

    We thank you for your positive review of our manuscript. We have now uploaded the EM densities to the EMDB (EMD-23403, EMD-23404, as noted at the end of our “Single Particle Analysis of CryoEM Data” Methods Section), but we have refrained from including our model structures as depositions to the PDB. Our hesitation is that the atomistic representations of our models do not reflect the relatively low resolution of their densities (9.5 Å and 11.5 Å), and we are concerned that accessors of the PDB deposition may overestimate the detail of these structures. Nevertheless, we agree that members of the community may benefit from hands-on examination of these models, so we have attached the PDBs as supplementary materials to this manuscript. In this way, readers can have open access to our model structures without implying a precision that is often inferred from a pdb deposition.

    We have decided against an expanded discussion of biological implications as we feel this might move us too far ‘out on a limb’. As relatively little is known about transcriptional regulation in archaea, we feel that adding speculations on top of speculation might be an overreach.

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  3. Reviewer #2 (Public Review):

    The manuscript "Archaeal chromatin 'slinkies' are inherently dynamic complexes with deflected DNA wrapping pathways" by Bowerman and colleagues describes a study of archaeasome dynamics combining molecular simulations, cryo-EM, and sedimentation velocity analytical ultracentrifugation. How chromatin evolved is a fundamental question in biology, marking a striking departure from the bacterial nucleoid. Indeed, ever since the first description of archaeal nucleosomes and histones HmfA/B (Sandman and Reeves mid-80s) from thermophilic archaea, this question has fascinated and puzzled the field.

    Recent work from the Luger lab figured out the organization of these archaeal chromatin fibers as a continuous loop structure. Here, the authors extend this question further. MD analyses show that Arc90 has two preferred states (closed and flexible ends), but the same 5T5K structure on 120 or 180 bp of DNA prefer a single state (closed). Sedimentation velocity analytical ultracentrifugation showed that Arc207 sediments slower than the H3 mononucleosome, implying that that Arc207 has a shape with higher anisotropy, resulting in excessive drag compared to a mononucleosome. Subsequently, high-resolution cryoEM showed that at least two distinct classes for Arc207 exist, where one class represents a 5-mer and another class represent a 7-mer. The latter has a unique shape in that the 7-mer forms an L-shape (or open clam) with a 3-mer hinging on a 4-mer.

    Overall, these data provide exciting structural insights into how archaeal chromatin is folded up at its basic unit level, which the authors describe as most fittingly as a "slinkie". Because so little is known about how nucleosomes evolved during the transition from archaea to eukaryotes, we found this interdisciplinary report well written and with compelling data, that will be of interest to the chromosome biology field at large. We suggest a minor revision in which a few technical points are addressed.

    Considerations:

    1. The cryoEM data showed two main groups of particles: 5-mer protecting 150 bp and a 7-mer protecting either 90bp or 120bp. A few times in the manuscript (both in the results and discussion section) the authors mention a 30-bp MNase digestion ladder is observed. The Mnase data should be included, as this provides evidence that the structures observed by cryoEM indeed represent physiological structures, especially if strong discrete bands are observed at 90, 120, and 150 bp.

    2. The two main classes found by cryoEM give the impression that adding dimers results in altered structures. The 7-mer shows an angled structure, which is interpreted as an open structure. The 5-mer shows a more uniform structure, which is interpreted as a closed structure. The former structure protects the full length of DNA on which HTkA histones were reconstituted, whereas the latter might be an incomplete reconstitution or a partially disassembled structure. It also raises the question if the length of the DNA is a limiting factor. What if HTkA was reconstituted on 170 bp or 307 bp instead? Would this in turn only permit the formation of the 5-mer on the 170 bp construct and two 5-mers on the 307 bp construct? The authors should consider addressing this point because the reconstitution might be constrained by the length of the DNA construct used. Indeed, a related topic might be AT content- what does archaeal DNA look like from the perspective of DNA sequence for chromatin (Jon Widom's group had a ChIPSeq paper on this a few years ago, just after his untimely passing).

    3. In the discussion the authors cite that in one archaeal species the Mg2+ concentration is ~120 mM, more than a magnitude greater than that tested in Figure 5. What happens to reconstituted archaeasomes at higher Mg+? This is relevant because in vivo, archaea are thought to have 10x the concentration of Mg+ (amongst other ions) relative to us humble eukaryotes who would probably die of kidney failure at those ionic concentrations. Indeed at high ionic conditions, eukaryotic chromatin can be made to precipitate out of solution (for e.g. 10mM Mg+, 3M NaCl). An AUC assay with higher Mg2+ concentrations seems a doable and physiologically relevant addition to the ms that would strengthen it. It is relevant to consider that in vivo structure in these halophilic and thermophilic organisms might be dependent on the concentration of various salts and temperature, it would be nice to read the authors' thoughts on this issue.

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  4. Reviewer #1 (Public Review):

    While I am not sufficiently qualified to comprehensively assess the molecular dynamics simulations, all interpretations seem careful and remain within the described limitations of the various metrics that the authors report.

    The experiments are well executed; the results are presented clearly and interpreted carefully. This is a rigorous and important biophysical study that provides a solid foundation for the investigation of archaeal genome biology. The authors' new findings raise interesting questions, and although addressing them is outside the scope of this study, the article would perhaps benefit from a more detailed discussion of the biological implications of the results. The manuscript does not indicate whether the cryo-EM maps and atomic models were deposited in the EMDB and PDB. I strongly encourage the authors to do that: it would add a lot of value not only for the readers of this study, but also for the wider structural biology community.

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  5. Evaluation Summary:

    In their manuscript titled "Archaeal chromatin 'slinkies' are inherently dynamic complexes with deflected DNA wrapping pathways", Bowerman et al. use an elegant combination of cryo-EM, analytical ultracentrifugation and molecular dynamics simulations to investigate the structure and dynamics of archaeal histone — DNA complexes, termed archaeasomes to distinguish them from eukaryotic nucleosomes. This study builds upon the crystal structure of an archaeasome and the functional analysis of its disruption recently published by the same group (Mattiroli et al, 2017) by analyzing the dynamics of this complex and discussing how these dynamics could relate to archaeal genome biology. How chromatin evolved is a fundamental question in biology, marking a striking departure from the bacterial nucleoid. This current manuscript describes a rigorous biophysical study that not only provides substantial new insights into archaeal genome biology but also raises intriguing questions for future study. This manuscript will therefore no doubt be of interest not only to the archaeal research community but also to the field of chromatin biology.

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

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