The most abundant protein in human urine, the glycoprotein uromodulin (UMOD; also Tamm-Horsfall protein, THP), is conserved throughout Mammalia and produced primarily in the epithelial cells lining the thick ascending limb (TAL) of the Henle loop in the kidney. There, UMOD is important for maintaining the water impermeable layer, regulating salt transport, and urinary concentration (Devuyst et al., 2017). Farther along the urinary tract, UMOD has been shown to act as a soluble adhesion antagonist against uropathogenic E. coli (UPEC) (Pak et al., 2001; Weiss et al., 2020).

UMOD precursors traffic through the secretory pathway in the assembly incompetent pro-UMOD form, where they become glycosylated at eight potential N-glycosylation sites and eventually attached at the apical membrane surface via their glycosylphosphatidylinositol (GPI)-anchored C-terminal pro-peptide (CTP) (Rindler et al., 1990; van Rooijen et al., 1999; Weiss et al., 2020). The protease hepsin then cleaves the CTP and UMOD assembles to homopolymeric filaments with an average length of 2.5 µm in the urine (Brunati et al., 2015; Porter and Tamm, 1955).

Recently, the general architecture of the intrinsically flexible UMOD filaments was uncovered via cryo-electron tomography (Weiss et al., 2020). The results, together with previous data, showed that UMOD assembles to a zigzag shaped, linear polymer, where the core of the filament is formed by its bipartite zona pellucida (ZP) module with the subdomains ZPN and ZPC. The N-terminal domains, epidermal growth factor-like (EGF) I‒III and the following cysteine-rich D8C domain, protrude as arms alternating from opposite sides of the filament, and the EGF IV domain connects the arms to the filament core (Figure 1A; Jovine et al., 2002; Schaeffer et al., 2009; Weiss et al., 2020). UMOD filaments encapsulate piliated uropathogens through multivalent binding via their N-glycans to the lectins at the tips of bacterial pili. This mechanism not only prevents pathogen adhesion to target glycans on epithelial cells of the host, but also facilitates pathogen aggregation and clearance by urine excretion (Weiss et al., 2020).

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Despite these insights, the exact structural organization of UMOD filaments remained unknown. We set out to solve the structure of the UMOD filament by single-particle cryo-EM. Isolated native UMOD filaments from a healthy individual appeared on the grid in two major views: a fishbone-like ‘front’ view with alternately protruding arms, and a zigzag ‘side’ view (Figure 1B). Marked conformational variation in both the filament core and the angles between the peripheral arms and the core was visible. Additionally, flexible N-glycans lining the filament core could be seen already in 2D class averages (Figure 1—figure supplement 1). This intrinsic flexibility and glycan display is a prerequisite of pathogen encapsulation by UMOD filaments, however, they considerably complicated the structure determination by single-particle cryo-EM, restricting high-resolution analysis to the filament core.

Ab initio 3D reconstruction of 723,000 particles was followed by extensive classification and focused refinement together with the non-uniform refinement strategy in cryoSPARC in order to obtain a reconstruction of the UMOD core at 3.5 Å (Figure 1—video 1). This high-resolution, unsymmetrized map was used to rigid body fit the individual UMOD ZP subdomains, ZPN and ZPC, from the previously published crystal structure of a recombinant, assembly incompetent pro-UMOD fragment (consisting of the EGF IV domain, ZP module, and CTP (termed rEGF-ZP-CTP); Bokhove et al., 2016; PDB: 4WRN). The resolution of the cryoSPARC map at the filament core allowed direct model building of the immunoglobulin-like (IG) ZP subdomain folds. The ZPN/ZPC asymmetric unit was then extended by rigid-body fitting within a lower resolution map (4.7 Å), refined in cisTEM, that contained more repeats. This fitting showed a helical architecture of the filament with a ~ 65 Å rise and ~180° twist, which is consistent with parameters obtained by sub-tomogram averaging (Weiss et al., 2020). From here, we were able to ascertain an unexpected, extended conformation of the ZP monomers in the filament core, wherein a long, 28-residue linker (UMOD residues L429‒F456) between the ZPN and ZPC subdomains of each ZP monomer spanned the ZPC from the previous module (ZPC_-1) and the ZPN from the subsequent module (ZPN₊₁) (Figure 1C–D). The core region including the linker is well resolved, showing clear sidechain map features (Figure 1E–F). The linker between ZPN and ZPC of the monomers spans ~103 Å and interacts tightly with the two intervening ZP subdomains (Figure 1D‒F, Figure 1—figure supplement 2). Moreover, Coulomb potentials for prominent N-glycans at N396 and N513 were observed and allowed visualization of the core pentasaccharide and disaccharide, respectively (Figure 1C,D,G).

The intrinsic flexibility of the UMOD fibers could be directly observed in the collected micrographs in the range of their curvatures (Figure 1B). Slight deviations in the helical rise along the chain and the intrinsic flexibility hampered particle classification and alignment and limited the resolution of the reconstructions. To analyze this flexibility, we measured the relative angles between subdomains after rigid-body docking into multiple, heterogeneous 3D class averages (Figure 1—figure supplement 3). Similar maximum angular changes of ~7 degrees were obtained at both unique interfaces between the ZP subdomains, indicating that both interfaces equally give rise to the flexibility of the filaments. Additionally, we utilized the 3D variability analysis from cryoSPARC (based on the principle component analysis (PCA); Punjani and Fleet, 2020), which showed a similar degree of filament bending (component 1) and an elongation of the filament along the z-axis (component 2) based on a relative rotation of ZP modules at the subdomain interfaces (Figure 1—video 2).

The elongated ZP linker establishes an intricate and extensively enchained scaffold for filament build-up from mature UMOD monomers. Reminiscent of donor strand complementation in subunit-subunit interactions of pili assembled via the chaperone-usher pathway (Waksman, 2017) or the more recently discovered type V pili (Shibata et al., 2020), formation of the UMOD filament involves a β-sheet complementation mechanism extending across enchained ZP modules (Figure 2A). Specifically, the extended linker between ZPN and ZPC in each monomer is comprised of three separate β-strands: Lβ₁, Lβ₂, and Lβ₃ (residues 430‒435, 438‒441, and 446‒452, respectively), where Lβ₁ and Lβ₂ of subunit n complement the fold of ZPC of subunit n‒1 (ZPC_-1), and Lβ₃ extends the IG-fold of ZPN of subunit n+1 (ZPN₊₁) (Figure 2B). The complementation site (CS) between Lβ₁, Lβ₂, and ZPC_-1 (denoted CS_A) is formed by the antiparallel insertion of Lβ₁ relative to the ZPC β_F strand, and the shorter, parallel complementation of the ZPC β_A’ strand by the Lβ₂ strand (Figure 2C). Lβ₃, the longest β-strand in the linker segment, binds parallel along the ZPN₊₁ β_G strand at complementation site B (CS_B) (Figure 2D).

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The inter-molecular interface between the ZPN subdomain of subunit n and the ZPC domain of the preceding subunit (ZPC_-1) (interface A, I_A), is formed by mostly hydrophobic residues from both subunits (Figure 2E), creating a buried surface area of ~2000 Ų (PISA server, Krissinel and Henrick, 2007). Specifically, residues Y402, Y427, and L429 from ZPN at I_A cover a prominent hydrophobic patch on the surface of the ZPC_-1, composed of L491, F499, F456, and L570 (Figure 2—figure supplement 1). The second unique intermolecular interface (I_B) along the elongated UMOD monomer is formed between ZPC_-1 and ZPN₊₁, and is spanned by the linker (Figure 2F). Surface charge complementary between the two ZP subdomains provides the basis for I_B, highlighted by the insertion of R415 on ZPN₊₁ into a negatively charged pocket on ZPC_-1 harboring D532. I_B is further stabilized by hydrophobic interactions between ZPC_-1 (F555, F553) ZPN₊₁ (I413, I414) and the linker (P441, V443) (Figure 2—figure supplement 1).

We utilized the Genome Aggregation Database (gnomAD) to parse missense variants in the UMOD ZP module detected in ~125,000 exome and ~15,000 whole-genome sequences from unrelated healthy individuals from various population studies (Karczewski et al., 2020). Given the selective pressure for high levels of functional UMOD filaments in the urine (Ghirotto et al., 2016), we reasoned that residues or regions of UMOD that are required for filament assembly should show less genetic variation in healthy individuals. Here, we found that both the linker segment and those residues important for creating I_A and I_B are depleted in genetic variants, realtive to the rest of the ZP module (Figure 2—figure supplement 2; Supplementary file 1A).

The previously reported structure of the pro-UMOD fragment rEGF-ZP-CTP, which crystallized as a homodimer in which only the ZPN subdomains interact (Bokhove et al., 2016), provides the nearest comparison for the transition from pro-UMOD (CTP intact) to mature UMOD in the filament. Figure 3 shows the major conformational differences between rEGF-ZP-CTP and the ZP module in the mature filament. The most prominent difference is the separation of ZPN and ZPC in the mature filament, where individual C^α atoms (e.g. ZPN residue S364) change their relative position by up to 95 Å (Figure 3A). In addition, formation of the 103 Å long linker segment in mature UMOD involves i) the excision of strand β_A from ZPC, which becomes the linker strand Lβ₃ in the mature ZP module, and ii) the unwinding and extension of the α-helical segment connecting ZPN and ZPC in pro-UMOD that forms linker strands Lβ₁ and Lβ₂ in the mature ZP module (Figure 3B; Figure 3—figure supplement 1). The overall folds of the ZPN and ZPC subdomains, however, remain the same (with C^α RMSD values of 2.0 and 1.2 Å, respectively). Notably, linker strand Lβ₂ occupies the same position in ZPC_‒1 as the β-strand segment (559‒605) from the CTP in rEGF-ZP-CTP. In the mature filament, the residues that constitute helix αEF of rEGF-ZP-CTP are extended and the involved aromatic residues undergo a rearrangement that allows binding of Lβ₂ (Figure 3—figure supplement 2). In addition, the insertion of the linker strand Lβ₁ into ZPC_-1 only becomes possible after the excision of its strand β_A, which blocks Lβ₁ binding in rEGF-ZP-CTP (Figure 3C–E). Overall, the binding pocket for the CTP β-strand in ZPC of rEGF-ZP-CTP is shorter than that accommodating Lβ₁ and Lβ₂ in the mature ZPC subdomain, causing a kink in the CTP peptide along the ZPC subdomain (Figure 3E). The difference observed in comparison of the ZPN domains is less dramatic; here, the same surface complemented by the rEGF-ZP-CTP strand β_G from the apposing ZPN in the crystal structure homodimer is fulfilled in the filament by the Lβ₃ strand from subunit n‒1 (Figure 3F,G).

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The cryo-EM structure of the UMOD filament core raises the intriguing question of how UMOD filaments assemble from pro-UMOD subunits attached to the cell surface. Figure 4 shows the simplest conceivable model of UMOD filament assembly. Assembly requires the release of mature UMOD monomers from the membrane by proteolytic removal of the GPI-anchored CTP by hepsin, thus making a ZPC module competent for binding the Lβ₃ strand of another UMOD. Assembly starts by binding of a pro-UMOD ZPN module to strands Lβ₁ and Lβ₂ in a neighboring pro-UMOD (step 1). Hepsin cleavage may then occur at the elongating filament (step 2). Each subsequent UMOD incorporation step (steps 3, 4, etc.) would then be characterized by the intercalation of the ZPN from an incoming pro-UMOD between the two ZPC modules of the filament, leading to a more stable binding of the incoming ZPN compared to that in step 1. As a final cleavage step to release the assembled filaments from the membrane, a yet unidentified hydrolase may act upon UMOD, as small amounts of the CTP could still be detected in MS/MS spectra after trypsin digestion of mature filaments (Figure 4—figure supplement 1). This UMOD assembly model is analogous to the recently proposed assembly mechanism of filamentous type V pili, in which assembly is linked with proteolytic release of pilus subunits from the outer bacterial membrane (Shibata et al., 2020). During the preparation of this manuscript, a related preprint article on the cryo-EM structure of the UMOD filament core was published (Stsiapanava et al., 2020). In said study, an alternative model of UMOD assembly was proposed, based on the assumption that assembly starts from membrane-bound pro-UMOD homodimers.

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The enchained β-sheet complementation mechanism observed in the UMOD filament core may also be valid for other proteins containing membrane-anchored ZP modules and undergoing filament formation. For instance, another human ZP protein, alpha-tectorin (TECTA), forms filaments creating the basis for the apical extracellular matrix (ECM) on the cochlear supporting cells and shares important features with UMOD: a highly similar linker region and a conserved, C-terminal protease cleavage site (Figure 4—figure supplement 2). Recently, Kim and colleagues proposed the ‘3D printing model’ for surface-tethered, TECTA-mediated ECM organization (Kim et al., 2019), which is in agreement with our proposed model of UMOD polymerization at the surface of TAL cells in the kidney tubule. Thus, the cryo-EM structure of the UMOD filament core might be representative for the core structure of multiple proteins with a C-terminal ZP module that become functional after polymerization.

Structures and maps presented in this paper have been deposited to the Protein Data Bank (PDB) and Electron Microscopy Data Base (EMDB) with the following accession codes: 3.5 Å cryoSPARC map EMD-11388, the UMOD AU model PDB ID: 6ZS5, elongated model of UMOD filament derived from cryoSPARC map PDB ID: 6ZYA, 4.7 Å cisTEM map EMD-11471.

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Thank you for submitting your article "The cryo-EM structure of the human uromodulin filament core reveals a unique assembly mechanism" for consideration by eLife. Your article has been reviewed by two peer reviewers, including Sjors HW Scheres as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by John Kuriyan as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Matthias Wolf (Reviewer #2).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

This paper describes cryo-EM reconstructions of the human uromodulin filament core. It follows on from a Science paper by the same group describing lower resolution tomography results and how UMOD filaments constitute a crucial component of the innate immune system against urinary tract infection. The presented structure is novel and unique. Interactions with biological filaments are important disease factors. The authors describe the first structure of the polymerized uromodulin filament core, providing experimental evidence for a β-strand complementing assembly mechanism similar to ones found in chaperone-usher or type V pili. The reviewers agreed that this paper is in principle suitable for eLife, but wondered whether the maps could be improved to provide stronger evidence for the proposed atomic model. The reviewers were of the opinion that statements regarding the speculative model should be toned down.

Major points:

1) Both reviewers carefully inspected the submitted cryo-EM reconstructions and models. The maps show pronounced radial blurring with increasing radius, making it difficult to interpret details off-axis. The interpretation of the map in terms of the proposed strand-exchanged model lies close to the limit of what these reviewers are comfortable with. Reviewer #1 was uncomfortable with some of the main-chain tracing decision made (See images 1, 2, 3 and 4); reviewer #2 pointed out that the model is consistent with other strand-exchanged structures, in particular the type V pili, thereby providing further confidence. In a revised version of the manuscript, the authors should do one, or both, of the following two options: 1) improve the quality of the map (see suggestions 2 and 3 below); 2) make additional supplementary figures that support the main-chain tracing in the current model, without hiding the limited quality of the map in the relevant regions.

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2) Paragraph six: No explanation is provided how the authors arrived at this symmetry parameter. How does the power spectrum look like of aligned averaged filament images? From the small raw micrograph image in Figure 1B it seems clear that the filament may not be strictly flat (rotation = 180 degrees, "snake on a table") but rather slightly twisted as seen in most of the filaments in this image (in particular the one pointing towards the letter B). While the authors attribute the limited quality of the map to the evident flexibility of the filaments, it could also stem from not accounting for the twist mentioned above. How would the maps look like, if the symmetry imposed used a helical parameter near 180 deg (i.e. 63A, 182.5 deg) or (63A, 177.5 deg)? These values are coarsely approximated from Figure 1B, assuming 90 deg twist over 5 scale bars (5*500A/63A = 40 subunits; 90deg/40 = 2.3deg).3) Alternatively, could the maps for specific regions where decisions about the main-chain trace are less clear be further improved by focused refinements on those regions?

4) The model in Figure 4 is based on a linear elongation model first proposed by Shibata et al. for Type V pili. At least, this work should be cited. The former also provided the first experimental validation of the donor strand exchange hypothesis proposed by Waksman et al. (please include citation). As compelling as their model is, the authors provide no evidence (short of detection of tryptic cleavage products by MS in Figure 4—figure supplement 1) for its individual steps. It is so speculative, that they feel compelled to offer an alternative version in the supplement (Figure 4—figure supplement 2A). Short of substantial evidence for this model, this hypothesis should either be greatly simplified and toned down, or left out altogether.

5) The authors use a new density-modification approach in phenix to produce a 4.2A map from the original 4.7A cisTEM map. It is unclear to me how density modification can lead to an increase in resolution. If the authors think this is real, they should explain why this is in a revised version. However, inspection of the map provided for review reveals that most β-strands are not resolved in the density-modified map, suggesting that the resolution is (indeed) still around 4.7A. If so, the 4.7A resolution number should be used.

6) The authors perform 3D classification and analyse heterogeneity in the data by RMSDs among pairs of fitted domains (Figure 1—figure supplement 2). RMSD is a very poor descriptor of the structural heterogeneity. Instead, the authors could calculate angles between subunits, or possibly do a PCA on the multiple structures, to describe the main types of structural variability.

7) Did the authors use Ramachandran restraints in their refinements? If so, this should be stated explicitly, as validation of Ramachandran outliers etc is no longer valid. If Ramachandran statistics are to be used for validation, they should be switched off during model refinement.

Major points:

1) Both reviewers carefully inspected the submitted cryo-EM reconstructions and models. The maps show pronounced radial blurring with increasing radius, making it difficult to interpret details off-axis. The interpretation of the map in terms of the proposed strand-exchanged model lies close to the limit of what these reviewers are comfortable with. Reviewer #1 was uncomfortable with some of the main-chain tracing decision made (See images 1, 2, 3 and 4); reviewer #2 pointed out that the model is consistent with other strand-exchanged structures, in particular the type V pili, thereby providing further confidence. In a revised version of the manuscript, the authors should do one, or both, of the following two options: 1) improve the quality of the map (see suggestions 2 and 3 below); 2) make additional supplementary figures that support the main-chain tracing in the current model, without hiding the limited quality of the map in the relevant regions.

We thank the reviewers for their careful inspection of the models and maps. As was pointed out, the cryo-EM maps indeed contain artifacts, which are caused by the high intrinsic flexibility of the UMOD filament, which leads to a higher resolution at the central axis and lower resolution at the periphery. Due to the flexibility of the filament, helical image reconstruction could not be applied and a number of approaches and software packages were used for solving the structure without applied symmetry (see also point 2). As a result, two best maps with complementary features were obtained: (i) a high resolution cryoSPARC map for the central asymmetric unit resulting from a focused refinement on the filament core. This map displays radial blurring with increased radius, likely due to the relatively small volume included in the focused refinement mask (corresponding to 5 ZP subdomains ~72kDa + glycosylation), (ii) A lower-resolution cisTEM map which includes more repeat units with better resolved periphery. In addition, several trials by signal subtraction and focused refinements using the Relion software were performed, however, those did not result in higher resolution maps likely due to the small size of the ZP subdomains (ZPN: 101 residues, ZPC: 128 residues) and the flexibility of subdomain interfaces (see comment 3 for details). The provided maps were of the best quality we could obtain after extensive optimization of masks and refinement parameters. Therefore, we followed the suggestion of the reviewers to support our conclusions by a better illustration of the lower-quality areas of the map (please see Author response images 1, 2 and 3).

Together with images 1, 2 and 3 we would like to make two clarifications:

We created two models (asymmetric unit (AU) and filament model), the latter of which was created by helically expanding the AU along the lower resolution cisTEM map and then refining it again against the 3.5 Å cryoSPARC map, in order to impose refinement restrains at the subunit interfaces. Each of the models has been manually cross-validated. We have added a sentence in the text clarifying that the model and the cryo-EM map should be interpreted on the chains A and D corresponding to the highest resolution of the map (central part) on which the refinement had been focused.

Specifically, to address reviewer 1’s comments about main chain tracing, we would like to reiterate that the AU model (comprised of a ZPN (chain A, residues 328–443), a ZPC (chain D, residues 444–584) and the spanning linker) was built into the center of the high-resolution cryoSPARC map.). The overall folds of these subdomains in the polymerized filament are very similar to the individual ZP subdomains from the previously published crystal structure (PDB: 4WRN; Bokhove et al., 2016, with RMSDs of 2.0 and 1.2 A for ZPN and ZPC, respectively. Additionally, we would like to clarify that the unique subdomain interfaces were built and subsequently refined only for the center of the volume where the map shows the least artifacts (Figure 1—figure supplement 2).

We now provide an additional figure (Figure 1—figure supplement 2) to illustrate the quality of the map in the central core showing the linker region (CS_A) with surrounding density. We display the Coulomb potential map as a mesh at two different contour levels, 18 σ (red) and 10 σ (blue), to show the continuity of the main chain density.

In Author response image 1 the arrow indicates the region of the map shown in the reviewer’s image; zoomed in views show the higher resolution, symmetry related, part of the map used for structure building.

Author response image 1

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Author response image 2

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Decision letter image 3 – Response: The image provided by the reviewer is a similar view as that Author response image 2; please see our response above.

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2) Paragraph six: No explanation is provided how the authors arrived at this symmetry parameter. How does the power spectrum look like of aligned averaged filament images? From the small raw micrograph image in Figure 1B it seems clear that the filament may not be strictly flat (rotation = 180 degrees, "snake on a table") but rather slightly twisted as seen in most of the filaments in this image (in particular the one pointing towards the letter B). While the authors attribute the limited quality of the map to the evident flexibility of the filaments, it could also stem from not accounting for the twist mentioned above. How would the maps look like, if the symmetry imposed used a helical parameter near 180 deg (i.e. 63A, 182.5 deg) or (63A, 177.5 deg)? These values are coarsely approximated from Figure 1B, assuming 90 deg twist over 5 scale bars (5*500A/63A = 40 subunits; 90deg/40 = 2.3deg).

We would like to reiterate that the 2D-classification of the picked UMOD particles revealed highly flexible and bent class-averages of a helical filament formed by repetitive subunits. Consequently, a single-particle analysis with no applied symmetry (C1 symmetry) was used. In order to emphasize this point, we have updated the main text. In initial trials, we attempted to apply helical processing in Relion but these refinements did not yield high resolution reconstructions, likely due to the flexibility of the filament.

Based on the reviewer’s comment we investigated the power spectrum of the 2D classes containing the straightest sections, but due to the small number of monomer repeats (4‒5 per class) it was not possible to interpret the power spectrum in the context of a helical structure, to perform the indexing, and to determine Bessel orders.

Upon further investigation based on the reviewers’ comments, we realized that the previously derived helical parameters had been determined from the high-resolution cryoSPARC map in the real space, using relion_helix_toolbox. However, due to the only 2 asymmetric units (an arm, ZPN and neighboring ZPC) in the map we decided to recalculate helical parameters in the same way using the cisTEM map, which includes ~4 asymmetric units. With a search range for helical rise of 60‒67 Å and twist of 175–185°, various cylindrical mask outer diameters (150–250 Å) and percentages of the cropped volume (0.3–0.6) we obtained values of a rise and twist to be ~65 Å and ~180° (with ~1% error), respectively. We updated the values in the manuscript and the Materials and methods section accordingly.

Also, we would like to draw the reviewers’ attention to the subtomogram averages that were previously calculated for direct structural analysis of the UMOD filament by cryo-electron tomography. The subtomogram averages of UMOD filaments show a helical rise of ~6.5 nm and twist of ~180° (Weiss et al., 2020) which is in agreement with the helical parameters obtained from the cisTEM map.

The appearance of different views within some individual filaments, as mentioned by the reviewer, we would attribute to local flexibility (see also answer to point 6).

3) Alternatively, could the maps for specific regions where decisions about the main-chain trace are less clear be further improved by focused refinements on those regions?

As mentioned in the reply to point 1, a focused refinement strategy (with and without signal subtraction; classification with and without alignments and using various masks) had been attempted. Due to high flexibility of the complex, small areas of focus (~12 kDa for individual subdomains of the thin filaments), the signal did not appear to be sufficient to drive refinements and perform better classification in the areas of interest. Taking under consideration our flexibility analysis of UMOD fibers, the resolution in focused refinements with a mask including more than a single subdomain was likely limited by subdomain movement. Also, we would like to point out the challenges related to the specimen preparation, which impose limits on the single-particle analysis of this complex: UMOD appeared to require a relatively thick ice layer to be located on the carbon support as an isolated, intact filament.

4) The model in figure 4 is based on a linear elongation model first proposed by Shibata et al. for Type V pili. At least, this work should be cited. The former also provided the first experimental validation of the donor strand exchange hypothesis proposed by Waksman et al. (please include citation). As compelling as their model is, the authors provide no evidence (short of detection of tryptic cleavage products by MS in Figure 4—figure supplement 1) for its individual steps. It is so speculative, that they feel compelled to offer an alternative version in the supplement (Figure 4—figure supplement 2A). Short of substantial evidence for this model, this hypothesis should either be greatly simplified and toned down, or left out altogether.

We would like to sincerely apologize for the oversight in not including the reference for Shibata et al., 2020. Due to a number of iterations in the manuscript preparation, it had been lost in the originally submitted version of our manuscript. Indeed, we had considered this work in our analysis from the methodological and functional point of view, since it describes a filamentous assembly that is highly analogous to UMOD filaments with respect to β-sheet complementation and proteolysis-dependent assembly competence. We followed the reviewers’ suggestion, curtailed the proposed assembly mechanism in Figure 4 and now emphasize the analogy to the mechanism of type V pilus assembly described by Shibata et al.

5) The authors use a new density-modification approach in phenix to produce a 4.2A map from the original 4.7A cisTEM map. It is unclear to me how density modification can lead to an increase in resolution. If the authors think this is real, they should explain why this is in a revised version. However, inspection of the map provided for review reveals that most β-strands are not resolved in the density-modified map, suggesting that the resolution is (indeed) still around 4.7A. If so, the 4.7A resolution number should be used.

We admit that the density modification approach in Phenix has not yet undergone peer review, and we removed the results of the density-modification in Phenix. Instead, we present the cisTEM map with standard sharpening procedures applied at 4.7 Å resolution. We amended the text and supplementary tables (Figure 1—figure supplement 1, Supplementary file 1B,D) accordingly.

6) The authors perform 3D classification and analyse heterogeneity in the data by RMSDs among pairs of fitted domains (Figure 1—figure supplement 2). RMSD is a very poor descriptor of the structural heterogeneity. Instead, the authors could calculate angles between subunits, or possibly do a PCA on the multiple structures, to describe the main types of structural variability.

We thank the reviewers for this comment and expanded our analysis accordingly. We have utilized the same fitting of individual ZP subdomains as rigid bodies into 3D classes and, instead of using RMSDs, measured the angle between the ZPC subdomains neighboring the central, aligned ZPN subdomain (see updated Figure 1—figure supplement 3). The angular movement of the subdomains of up to 7 degrees, provides a basis to describe filament bending (the angular movement between subdomains propagates along the filament and severely complicates reconstruction of longer filament segments).

Additionally, as suggested, we performed the PCA-based 3D-variablity analysis in the cryoSPARC software. The first component shows a bending motion of the filament similar to our flexibility analysis of 3D classes. We prepared a new figure indicating the main variability components and added a description to the main text (Figure 1—video 2).

7) Did the authors use Ramachandran restraints in their refinements? If so, this should be stated explicitly, as validation of Ramachandran outliers etc is no longer valid. If Ramachandran statistics are to be used for validation, they should be switched off during model refinement.

We have updated the Materials and methods section and the Supplementary file 1B and now explicitly state that the standard settings for refinement in RosettaCM and phenix.real_space_refine were used, including Ramachandran restraints.

Author details

1. Jessica J Stanisich

   Institute of Molecular Biology & Biophysics, ETH Zurich, Zurich, Switzerland

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing

   Contributed equally with

   Dawid S Zyla

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2702-8092

2. Dawid S Zyla

   Institute of Molecular Biology & Biophysics, ETH Zurich, Zurich, Switzerland

   Contribution

   Conceptualization, Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing

   Contributed equally with

   Jessica J Stanisich

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8471-469X

3. Pavel Afanasyev

   Cryo-​EM Knowledge Hub (CEMK), ETH Zurich, Zurich, Switzerland

   Contribution

   Conceptualization, Data curation, Software, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Project administration

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6353-6895

4. Jingwei Xu

   Institute of Molecular Biology & Biophysics, ETH Zurich, Zurich, Switzerland

   Contribution

   Data curation, Validation, Methodology

   Competing interests

   No competing interests declared

5. Anne Kipp

   Institute of Physiology, University of Zurich, Zurich, Switzerland

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization

   Competing interests

   No competing interests declared

6. Eric Olinger

   Translational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Central Parkway, Newcastle upon Tyne, United Kingdom

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1178-7980

7. Olivier Devuyst

   1. Institute of Physiology, University of Zurich, Zurich, Switzerland
   2. Division of Nephrology, UCLouvain Medical School, Brussels, Belgium

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3744-4767

8. Martin Pilhofer

   Institute of Molecular Biology & Biophysics, ETH Zurich, Zurich, Switzerland

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3649-3340

9. Daniel Boehringer

   Cryo-​EM Knowledge Hub (CEMK), ETH Zurich, Zurich, Switzerland

   Contribution

   Conceptualization, Resources, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

   Competing interests

   No competing interests declared

10. Rudi Glockshuber

    Institute of Molecular Biology & Biophysics, ETH Zurich, Zurich, Switzerland

    Contribution

    Conceptualization, Resources, Supervision, Funding acquisition, Investigation, Writing - original draft, Project administration, Writing - review and editing

    For correspondence

    rudi@mol.biol.ethz.ch

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-3320-3843

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