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  1. Evaluation Summary:

    This work combines 2D and 3D cryo-electron microscopy to show that cellular keratin intermediate filaments have heterogenous diameter, protofilament number and protofilament arrangement. This demonstrates the challenge for future high resolution structure determination of these essential filaments as well as providing the basis for understanding how this heterogeneity facilitates their function.

    (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.)

  2. Reviewer 1 (Public Review):

    This study reports on the cryoEM/cryoET determination of keratin filament structure in mouse keratinocytes genetically engineered to express K5 and K14 as the only keratin pairing. As described the study feels thorough and well-executed. This data is a first in the field of keratin research, and provides a much needed framework to understand, the structure, properties, and roles of keratin filaments. The study should be of high interest to the readership.

    Some of the findings of the study confirm and amplify what was already known about keratin filaments in general and/or K5-K14 filaments in particular - e.g., diameter, fibrillar substructure, persistence length, and structural heterogeneity. Other than the important fact that the data reported in this paper pertains to native filaments in a live cellular setting, the main finding of interest is the substructure of the filaments, given six "protofilaments" and the description of a central dense core (an unanticipated finding). Also of interest is the data about the classes of axial repeat patterns, which may represent different physiological states.

  3. Reviewer #2 (Public Review):

    In this study, Weber et al. study the structure of keratin intermediate filaments (KIFs) in detergent extracted 'ghost' cells using cryo-electron microscopy. This allows their observation after expression, post-translational modification and assembly in their native environment. To limit the compositional heterogeneity of the KIFs, the authors generate a cell line which contains only K5/K14 filaments. They analyse the structure of straight KIFs using 2D classification of cryo-EM images, showing they vary in diameter and protofilament helicity. They further characterize variations in the helical repeat distance and assign this to changes the angle of observation of the filaments along their long axis. Reconstruction of individual filaments from the classified particles shows they can contain numerous transitions in width and helical patterning. Next, they use cryo-electron tomography to show that KIFs drastically change Z-height in cells. This allows observation of cross sections of the filaments and reveals there is density in their center. In some cases, six protofilaments are clearly visible in the cross sections but there also appears to be heterogeneity in their number.

    Overall, the structural heterogeneity of cellular KIFs is clear from this data and the work is very well executed and presented. A nice contrast to other cytoskeletal filaments is provided, as they are able to solve the structure of actin filaments using the same pipeline to 6.1 Angstrom resolution. The combination of 2D classification and analysis of 3D cryo-electron tomograms is well utilised to show how these flexible filaments can change in diameter and have different protofilament architecture. Currently, it is not clear whether the variable helical repeat distance observed is a true feature of the filaments or due to filament tilting. This requires clarification but does not impact the main conclusions of the manuscript. This work demonstrates the challenge of understanding the details of how KIFs are built at high resolution. In addition, how the observed heterogeneity is modulated, for example in different parts of the cell or under different conditions, could now be addressed.