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  1. Evaluation Summary:

    Lövestam et al. report 76 cryo-EM structures of in vitro assembled recombinant tau filaments, including 27 previously unobserved ones. Together with the recent paper from Scheres and Goedert research groups, the structure-based knowledge of amyloid assembly will be boosted several fold. Most importantly, a few in vitro conditions were found to replicate the amyloid structures from both Alzheimer's disease and chronic traumatic encephalopathy. Those findings will open up new avenues to quickly screen compounds that inhibit filament formation under in vitro conditions, as well as the (self-)assembly process of amyloid fibrils.

    (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, Reviewer #3 and Reviewer #4 agreed to share their name with the authors.)

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  2. Reviewer #1 (Public Review):

    Scheres and colleagues report 76 cryo-EM structures of recombinant tau filaments assembled in vitro. This is a scientific tour-de-force, and will provide an immense database that can be used by everyone working in the amyloid field. When this knowledge is combined with the structure of tau filaments in vivo, it will help shape the design of laboratory experiments in the future to generate the conditions to replicate the in vivo forms in vitro.

    For one filament solved at 1.8 Å resolution, they determined the hand of these polymers from the carbonyl oxygens. But I am confused and puzzled about how the hand was determined or chosen for the rest. The reason that this is important is because it is stated: "Whereas all previously described tau filaments had a left-handed twist, phosphoserine-induced filaments were right-handed." Is it actually true that all previously described tau filaments had a left-handed twist, or was that assumed in many papers? I actually asked the authors of two recent papers in the amyloid field about what experimental evidence they had for stating that their filaments were left-handed. In both cases, they replied that this was an assumption based upon the literature, and it was a mistake to never state this in the paper (or, in one case, to actually imply that the filaments were observed to be left-handed). While I had thought that one might require almost near-atomic resolution to see the hand directly, a recent PNAS paper on a largely beta structure (10.1073/pnas.2120346119) showed that the hand could be determined at 2.5 Å resolution. I therefore think that it would help the amyloid field greatly to explicitly state in all cases when the hand has been determined, when it has been assumed, and test what resolution might be required to have confidence in describing the hand.

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  3. Reviewer #2 (Public Review):

    Lovestam et al. report the results of cryo-EM studies of tau protein fibrils grown in vitro under a variety of conditions. One goal of this work is to identify conditions that yield the same tau fibril polymorphs that the MRC group has identified in earlier studies of fibrils that were extracted from AD, CTE, Pick's disease, and CBD. They find that certain conditions, influenced by agitation rate during fibril growth, salt concentration, the nature of cations, and crowding agents, and truncation of the tau construct, do yield fibrils that are identical to (or at least very similar in their core structures) to fibrils from AD and CTE tissue samples.

    This work may represent the first example of the use of cryo-EM-based structures to evaluate the results of a broad screening of growth conditions. As such, this work demonstrates the value of "high-throughput" cryo-EM.

    This work also shows that tau polymorphs found in brain tissue do not necessarily depend on the presence of brain-specific cofactors or brain-specific conditions, as the same polymorphs can in some cases be created in the complete absence of brain material and without seeding with brain-derived seeds.

    This work also provides further examples of the amazing diversity of amyloid fibril structures that can arise from a single amino acid sequence and the sensitivity of polymorphism to subtle variations in growth conditions (e.g., agitation rate), as was first shown by other for amyloid-beta fibrils. Additionally, the authors find polymorphs with three-fold symmetry about the fibril growth axis (e.g., their "10a" and "15a" structures) as well as two-fold symmetric polymorphs (e.g., "4a","8a", "8b", etc.). This is interesting, because 40-residue amyloid-beta fibrils have also been shown to exhibit two-fold and three-fold symmetric polymorphs.

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  4. Reviewer #3 (Public Review):

    In this article, Lövestam et al. reported 76 cryo-EM structures of in vitro assembled recombinant tau filaments, including 27 previously unobserved ones. Together with the recent Science paper from Scheres and Goedert research groups, the structure-based knowledge of amyloid assembly will be boosted several fold. Most importantly, a few in vitro conditions were found to replicate the amyloid structures from both Alzheimer's disease and chronic traumatic encephalopathy. Those findings will open up new avenues to quickly screen compounds that inhibit filament formation under in vitro conditions, as well as the (self-)assembly process of amyloid fibrils. Congratulations on this beautiful body of work!

    Here is a minor point:

    It seems all helical twists per ~4.8Å rise are assumed to be left-handed, meaning the twist of β-sheets was assumed left-handed. I wonder whether AFM or Tomography was ever used to determine the hand of the filaments experimentally. Some filaments reached to high resolution, such as 1.8 Å. The author stated: "At this resolution, the absolute handedness of the filament was obvious from the position of the main-chain carbonyl oxygens. Whereas all previously described tau filaments had a left-handed twist, phosphoserine-induced filaments were right-handed". This is nice, and it will further benefit the field if the authors can use their best filaments, let's say ten in terms of resolution, mirror the maps, and build atomic models into those mirror maps. Then within the original and mirror models, they can show whether β-sheet hydrogen bonds get worse in the mirrored map. We recently found this can be pretty indicative of the correct hand for cross-β filaments that go to ~3Å or better. It would be much better for the field to understand the hand of cross-β filaments rather than assuming it.

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  5. Reviewer #4 (Public Review):

    The manuscript describes the investigation cryoEM structures formed by a major fragment of tau in vitro under a range of different assembly conditions. The work then goes further and examines the effect on amyloidogenic potential and structure of lengthening or shortening the sequence, and of introducing phoshorylation mimicking substitutions. This manuscript reports the cryoEM structures of 76 polymorphs formed by tau peptides. Of these, some a very similar or identical to those extracted from brains of individuals with Alzheimer's disease or chronic encepthalopathy, while others are polymorphs not previously solved. This paper shows the huge range of possible structures formed by the tau peptide. Furthermore, the range of conditions tested explores the influence of environment on resulting structures, showing the importance of salts. The influence of the N and C termini of full length tau is examined and only when phosphorylation mimics are included in the sequence, are some longer fragments of tau able to self-assemble.

    The extensive work shown here addresses some fundamental questions regarding tau self-assembly in disease and provides a huge number of structures. This work can be used to extend our knowledge of how tau is involved in disease pathology and causation and how different polymorphs are associated with very different tauopathies.

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