Cryo-EM structures of plant Augmin reveal coiled-coil assembly, antiparallel dimerization, and NEDD1 binding

Domain: physics
Published: 2025-12-12
Editorial outcome: major_revision

Abstract
Microtubule (MT) branch nucleation requires Augmin and NEDD1 proteins, which recruit and activate the gamma-tubulin ring complex (γ-TuRC). Augmin is a fork-shaped assembly of eight coiled-coil subunits, while NEDD1 is a β-propeller protein bridging MTs, Augmin, and γ-TuRC. We reconstitute Arabidopsis thaliana Augmin assemblies and determine 3.7-7.3-Å cryo-EM structures of its V-junction and extended regions using crosslinking mass spectrometry. These structures reveal a complete plant Augmin model showing multi-coiled-coil interfaces stabilizing its 40-nm hetero-octameric fork architecture. The dual calponin homology (CH) domains at the V-junction terminus adopt open and closed conformations for MT binding. A 12-Å cryo-EM structure shows Augmin undergoes anti-parallel dimerization through conserved surfaces on its extended region. We determine the NEDD1 β-propeller structure with Augmin, revealing direct binding inside the V-junction that enhances dimerization. Direct coupling and evolutionary analyses identify co-varying residue pairs validating the eight-subunit model and NEDD1 interface. Cooperativity between dual CH domains and NEDD1 binding may regulate V-junction binding to MT lattices. This V-shaped dual binding anchors Augmin along MTs, creating platforms for γ-TuRC recruitment and branched MT nucleation.

Introduction
Microtubule (MT) nucleation is essential for organizing the cytoskeletal networks1,2. Across eukaryotes, MT nucleation requires the highly conserved cone-shaped γ-tubulin ring complex (γ-TuRC), which nucleates nascent MTs by templating their tube-like, thirteen protofilament organization3,4. The γ-TuRCs nucleate MTs either from MT organizing centers or as branches alongside polymerized MTs1,2. In animal cells, the γ-TuRCs localize to centrosomes and nucleate most MTs during interphase, leading to a centralized cellular MT network with polymerizing dynamic MT plus-ends extending to the cell periphery, while the MT minus ends are anchored to the γ-TuRC-anchored centrosome1,2. In contrast, in plant cells, which lack centrosomes, γ-TuRCs often localize along existing MTs and nucleate MT branches to form a near parallel cortical MT network5,6. In mitotic cells for both plants and animals, γ-TuRCs are recruited to bind along mitotic spindle MTs and produce parallel MTs extending towards chromosomes in the mitotic spindle7. Augmin is required for centrosome-independent γ-TuRC-activated MT branch nucleation in mitosis, leading parallel MTs to form bipolar mitotic spindles. The activities of Augmin and Neural precursor cell expressed, developmentally downregulated 1 (NEDD1) are necessary for aligning and segregating chromosomes during cell division, and the defects in its eight subunits lead to long and thin mitotic spindles7,8. Drosophila melanogaster RNAi screens for mitotic phenotypes identified a subset of the eight Augmin subunits as essential for γ-tubulin association with spindle MTs and producing parallel MT arrays in mitosis9. In contrast to the most mitotic functions of Augmin in animals, plant Augmin promotes γ-TuRC MT-branch nucleation in both interphase and mitosis5–7.

Recombinant γ-TuRCs are weak MT nucleators, and conserved factors recruit, anchor, and activate them at centrosomes or along dynamic MTs10–12. Augmin is among the most conserved γ-TuRC-associated factors across plants and animals13. The metazoan Augmin complex consists of eight distinct coiled-coil subunits, termed HAUS 1,2,3,4,5,6,7,814. Eight equivalent plant Augmin subunits, named AUG 1,2,3,4,5,6,7,8, were identified in Arabidopsis thaliana, revealing their essential function for MT-branch nucleation during both interphase and mitosis15–17. Plant genomes, however, include eight distinct AUG8 paralogs, suggesting a diversity of Augmin functions in mediating MT branch nucleation in plant cells7,16. In interphase, branched MTs emerge on average at 40° (incident angle) from the polymerized MTs, whereas in mitosis, branched MTs emerge at 10°, leading to a mostly parallel MT array, suggesting a diversity of Augmins with unique mitotic and interphase functions specified by distinct AUG8 subunits7,16.

In addition to Augmin, other MT associated proteins (MAP) regulate Augmin MT association. TPX2 has been shown to recruit Augmin to MTs in Xenopus laveis extracts and is conserved in both plants and animals18; however, it is totally dispensable in plants19. In contrast, NEDD1, also termed GCP-WD, is a highly conserved MAP across plants and animals, and defects in it critically impacts MT nucleation function in both systems18,20. NEDD1 consists of a WD40 β-propeller domain (termed NEDD1-WD β-propeller) and a C-terminal helical coiled-coil20,21. Human mutations in NEDD1 or Augmin are linked to neurological disorders and are dysregulated in neural precursor cells22–24. Similarly, defects in NEDD1 and Augmin are lethal in plants, suggesting conserved roles and mechanisms in MT nucleation7,25. Live cell imaging in Drosophila cells during anaphase show that Augmins bind to MTs, followed by γ-TuRC recruitment to nucleated daughter dynamic MTs26. A recent study suggested NEDD1 recruits Augmin to bind to MTs and then recruit γ-TuRC to activate MT branch nucleation20. Furthermore, these studies suggest that Augmins oligomerize upon binding MTs prior to recruiting γ-TuRC20. However, the nature of the oligomerization of Augmin assemblies or their structure remain unknown. The structural mechanisms of Augmin and NEDD1 in regulating γ-TuRC activation along dynamic MTs have also mostly remained poorly understood, in part, due to the lack of reconstitution studies of these systems.

Augmin assemblies are hetero-octamers composed of 30-nm extended region that end with a 10-nm wide V-shaped junction (V-junction), resembling the shape of a “tuning fork”. Biochemical studies suggest the Augmin V-junction binds MTs via its conserved dual-headed CH MT binding domain in HAUS 6,7 (AUG6,7) and positively charged disordered N-terminal region of Haus 8 (AUG8), all of which reside at the tip of the long arm of the V-junction14. However, it is not clear how the second end of the V-junction stabilizes Augmin binding to MTs. The Augmin dual CH-domains have been compared to the well-studied NDC80/Nuf2 kinetochore complex, which contains a similar dual set of MT binding CH domains27,28. The conformation of the dual CH-domains upon MT binding remains unknown. It also remains unknown how Augmins anchor along MTs via their V-junctions and recruit γ-TuRC via their extended region. Recent reports of multiple low-resolution Augmin cryo-EM structures, in combination with AlphaFold2/ColabFold models, have led to structural models for the eight coiled-coil assembly, suggesting a general view of the hetero-octameric organization29–31. However, even among the clearest of these cryo-EM maps, low-resolution α-helical density is observed, and most of the coiled-coil assembly interactions were inferred by placing AlphaFold2 models into the low-resolution cryo-EM density maps. Difficulties with studying the structures of Augmin structures stem from their highly elongated shape and flexibility, hindering crystallographic or cryo-EM structure determination studies.

Our study presents a comprehensive structural and biochemical analysis of the Augmin assembly. By reconstituting recombinant A. thaliana Augmin hetero-octamers and hetero-tetramers assemblies, we utilized crosslinking mass spectrometry (XL-MS) and single particle Cryo-EM to determine structures for different regions of the Augmin assembly, leading to a near-complete de novo Augmin model. Our structural analysis reveals insights into the dynamic flexibility and states of Augmins, such as the conformation of AUG6,7 dual CH-domains at the tip of the V-junction, which adopt both splayed and packed states. Furthermore, we observed that Augmins undergo anti-parallel dimerization, mediated by conserved interfaces along their extended regions. This organization was visualized in a 12-Å cryo-EM structure of the Augmin dimer assembly, highlighting the spatial arrangement and transitions of the extended domain. Our findings also demonstrate that NEDD1 WD β-propeller binding to Augmins requires their V-junction region, and it enhances Augmin dimerization. The 10-Å cryo-EM structure of the Augmin-NEDD1 β-propeller revealed its binding site inside the V-junction. We present direct coupling and evolutionary analyses that reveal pairs of co-varying residues, validating our eight-subunit Augmin model and its NEDD1 β-propeller interface site. Our results lead us to a structural model where Augmin binding to MTs is stabilized by AUG6,7,8 and the NEDD1 β-propeller, positioned on different ends of the V-junction to anchor Augmin along multiple MT-protofilaments. This arrangement likely creates a platform for anchoring γ-TuRC, thereby facilitating MT branch nucleation. Our work significantly advances the understanding of Augmin’s role in MT dynamics, providing detailed molecular insights into its interactions and structural organization.

Results
Biochemical reconstitution of plant Augmin assemblies
To purify A. thaliana Augmin assemblies, coding regions for AUG1,2,3,4,5,6,7,8 subunits were assembled into polycistronic bacterial expression vectors (Supplementary Fig. 1). The AUG1, AUG3, AUG4, and AUG5 subunits consist mostly of highly conserved α-helices, while AUG2, AUG6, AUG7, and AUG8 were relatively less conserved15. The MT binding region of AUG8 is highly divergent and predicted to be disordered or unstructured and was thus excluded from expression (Fig. 1A). To overcome many rare Leu and Arg plant codons, codon-optimized AUG2,7,8 sequences were used for bacterial expression. We generated three polycistronic co-expression vectors consisting of either eight subunits (AUG1,2,3,4,5,6,7,8) or four subunits (AUG1,3,4,5 or AUG2,6,7,8) (Supplementary Fig. 1). Polycistronic expression led to soluble assemblies of AUG1,2,3,4,5,6,7,8 and AUG1,3,4,5 (Supplementary Fig. 1). However, AUG2,6,7,8 assembly were unstable and could not be purified, suggesting that AUG2,6,7,8 require the AUG1,3,4,5 for their solubility and can only be studied as a part of the full Augmin (AUG1,2,3,4,5,6,7,8). Mass spectrometry confirms each Augmin subunits is purified in the assembly (Supplementary Fig. 1E). The AUG6 C-terminal region is prone to degradation with multiple polypeptide bands (Supplementary Fig. 1C). Mass spectrometry also identified several low amounts of bacterial contaminating proteins, suggesting that they copurify with Augmin assemblies through multiple steps (see below; Supplementary Fig. 12). We measured the masses of Augmin assemblies using size exclusion chromatography with multi-angle light scattering (SEC-MALS) and mass photometry (MP) showing AUG1,3,4,5 are hetero-tetramers with a mass of 250–280-kDa mass, and the AUG1,2,3,4,5,6,7,8 assemblies forma hetero-octamers with a mass of ~380–420 kDa mass (Fig. 1B, D and Supplementary Fig. 1D, H). The measured masses suggest a stoichiometry of one Augmin subunit per Augmin assembly, consistent with human Augmin14. Although the plant AUG1,2,3,4,5,6,7,8 hetero-octamers are stable, changes in ionic strength led to their destabilization through a cascade of AUG2,6,7,8 dissociation, likely caused by the degradation or dissociation of AUG6, resulting a mixture of hetero-octamers (AUG1,2,3,4,5,6,7,8) and hetero-tetramers (AUG1,3,4,5) over time (see below Supplementary Fig. 7F).

Cryo-EM of Augmin reveals its architecture and dimerization
We collected cryo-EM datasets for hetero-octameric (AUG1,2,3,4,5,6,7,8) and Hetero-tetrameric (AUG1,3,4,5) Augmin assemblies (Supplementary Fig. 2 and Fig. 1E). 2D-class averages show that the AUG1,2,3,4,5,6,7,8 particles have a characteristic 40-nm tuning-fork shape, similar to previously determined structures of Augmin (Fig. 1E, top panel)29–31. 2D-class averages of AUG1,2,3,4,5,6,7,8 particles reveal a 30-nm extended region with a wide center, an narrow leg-like extension, and a foot-likedensity at one end, which closely resembles the shape of the AUG1,3,4,5 particles (see below). The other end forms a V-junction shape, leading to a second arm with a globular end (Fig. 1E, second panel). The V-junction consistently possesses a high electron density spot. Mass spectrometry analysis is suggestive that this interactor is RplB—a bacterial β-barrel ribosomal protein that crosslinks to AUG5 in this region and may remain associated with rRNA which could potentially explain the high electron intensity of this spot (see below Supplementary Fig. 12 and Fig. 1E). In many cases, the 2D-class averages show the crescent-shaped V-junction without the 24-nm extended region, suggesting this region may be either broken off or out of focus in those images (Fig. 1E, second panel). 2D-classes of 40-nm particles reveal dual extended region densities with a single V-junction (Fig. 1E, third panel). Finally, 2D-classes of 65-nm particles display dimeric, C2-symmetric Augmin particles with dual anti-parallel tuning forks, indicating that Augmin dimers form in an anti-parallel manner via their respective extended regions (Fig. 1E, fourth panel). These diverse types of 2D-class averages suggest that these AUG1,2,3,4,5,6,7,8 particles have a propensity for dimerization, with some particles potentially losing the V-junction, which aligns with the biochemical interpretation of Augmin dissociating into sub-complexes. However, nearly half of the AUG1,2,3,4,5,6,7,8 particles are missing the extended region (Fig. 1E, second panel).

The 2D-class averages for the AUG1,3,4,5 assemblies show ~24-nm elongated filament-like particles with clear secondary structural elements. These particles have a wide profile near their center, an narrow leg-like density connected to a foot-like domain at one end, and an elongated short extension at the other end (Fig. 1E, bottom panel). The 2D-classes of AUG1,3,4,5 particles exhibit similar features to the lower extended region of the AUG1,2,3,4,5,6,7,8 particles (compare Fig. 1E, top and bottom panels). These particles are visible from multiple angles, including end-on views (Fig. 1E, bottom panel; Supplementary Fig. 2). These observations suggest that the foot region is mobile and flexible in the cryo-EM images, despite the more ordered structure of the extended region.

Single particle cryo-EM structures of the full Augmin assembly
2D-class averages for the hetero-octameric Augmin particles (AUG1,2,3,4,5,6,7,8) show a mixture of mostly side and top views (Fig. 1E, panels I and II). As a result of the low signal to noise due to the large box sizes and extensive flexibility, we were only able to determine a consensus 9-Å Augmin structure (Supplementary Fig. 2). This reconstruction shows characteristic features of previously seen for the Augmin (Supplementary Fig. 2, lower left)29–31. Heterogeneity analysis allowed us to determine a low-resolution reconstruction for the Augmin particles with a second extended section, termed Augmin 1.5, revealing an incomplete Augmin dimer particle (Supplementary Fig. 2, lower middle). We also isolated the Augmin dimer particles and applied C2 symmetry, leading to a 12-Å resolution reconstruction of the central core dimer interface, termed the Augmin dimer (Supplementary Fig. 2, lower right; see below).

To determine the single particle structures of the Augmin’s V-junction-stem region with greater clarity, we combined all particles with the Augmin V-junction by recentering and re-extracting this region from the AUG1,2,3,4,5,6,7,8 datasets, and processing them as described in the scheme presented in Supplementary Fig. 3. The flexibility analyses revealed variable rotation of up to 20° in the long arm of the Augmin V-junction (Supplementary Figs. 3 and 7A–C). We determined two reconstructions for this V-junction-stem region: (1) a 7.3-Å resolution reconstruction representing a state in which the dimeric CH domains are tightly packed, which we termed the “closed state” (Fig. 2A, B, right and middle panel; Supplementary Fig. 3, lower left). (2) a 10-Å resolution reconstruction representing a state in which the CH domains are splayed apart, which we termed the “open state” (Fig. 2A, right panel). In the open state, the bow density rotates downward by ~15° with respect to the base of the V-junction. Vectorial comparisons of the residue-to-residue movements for models for the two states show that AUG2,6,7,8 moves upward, while the AUG3,5 in the base moves upward (Supplementary Fig. 7C). The 7.3-Å cryo-EM map of the closed state shows clear helical connectivity in the base of the V-junction, and that the closed dual-pronged packed CH domains are visible at the end of the V-junction (Fig. 2B, left and middle panel; Supplementary Fig. 5D–I). In contrast, the 10-Å cryo-EM map of the open state shows the rotation of the V-junction and the opening of the dual-pronged CH-domains (Fig. 2B, right panel; Supplementary Fig. 6; see below). These structures reveal the transitions of the V-junction and the globular head domain of AUG6,7 suggesting that their dual CH-domains undergo an open and close transition (Supplementary Fig. 6H–J).

We determined a 3.7-Å cryo-EM structure of the hetero-tetrameric Augmin (AUG1,3,4,5) using the scheme presented in Supplementary Fig. 4. Our biochemical reconstitution of this minimal Augmin assembly led to a high resolution cryo-EM structure that reveals clear helical density and side chain interactions, previously not observed in the metazoan Augmin structures (Fig. 2C and Supplementary Fig. 5J–L)29–31. The maps reveal the detailed folding organization of AUG1,3,4,5 subunits in the extended region of Augmin. The end of the AUG1,3,4,5 particle was flexible, and we thus utilized a combination of 3D-variability, flexibility, and local refinement to obtain a 6-Å reconstruction for this region (Supplementary Figs. 4 and 5M). The previous Augmin structures have not resolved the terminal extended region, likely due to its flexibility, coupled with its consistently inaccurate prediction by AlphaFold2 models29–31. The helical features of this tripod-shaped terminal domain are clear, allowing for the accurate placement of and morphing of AlphaFold models (Fig. 2D and Supplementary Fig. 5B, C, J–M).

Using the overlap between the cryo-EM density maps of the 7.3–3.7 Å resolution of the V-junction stem and the extended region and their fit into the consensus 10-Å Augmin particle cryo-EM structure, we are able to fully map helical densities across the 40-nm Augmin hetero-octamer particle (Fig. 2B, D, E, Supplementary Fig. 5A, B, and Supplementary Movie 1). A globular density, representing a bright spot residing inside the V-junction, potentially corresponds to the ribosomal protein, RplB. Mass spectrometry on the Augmin pulldowns shows RplB to be an abundant contaminant with 518 peptide-spectrum matches, and moreover RplB crosslinks to AUG5 in this region (see below; Supplementary Fig. 12). A β-barrel domain was placed in this density but was excluded from our model building and interpretation (Fig. 1E, fifth panel; Supplementary Fig. 5B, G). The RplB β-barrel proteins likely occupy a “sticky” binding site in Augmin, substituting for a missing binding partner in the bacterial expression system (see below). Hence, our single particle cryo-EM structures lead to full density maps of all structured regions of the Augmin particle, allowing us to build a near-complete structural model for the Augmin hetero-octamer (Fig. 2E and Supplementary Movie 1).

Completed de novo model of Augmin reveals intertwined coiled-coils
Using our overlapping cryo-EM maps for the Augmin V-junction-stem and the extended regional, we were able to generate a de novo model of the Augmin hetero-octamer (Fig. 2E, Supplementary Fig. 5B, C, and Supplementary Movie 1). The extended region is a 30nm long assembly with a wide multi-helical region in its center, termed the “belly”, connected to a narrow four-helical bundle below, termed the “leg”, which terminates into three-prong multi-helical density, termed the “tripod” (Fig. 2E and Supplementary Movie 1). On the other end of the belly region, a four-helical bundle curved structure, we termed the “stem”, extends toward the V-junction leading to a nexus point, we term the “joint” (Fig. 2E and Supplementary Movie 1). A short extension in line with the stem extends above the joint forming one arm of the “V”. The other arm of the V is composed of a long four helical bundle AUG2,6,7,8, termed the “bow”, which ends with a globular region, termed the “head”, composed of the AUG6,7 CH domain dimer (Fig. 2E and Supplementary Movie 1).

Our Augmin model explains the multi-subunit coiled-coil helical interactions that stabilize the hetero-octamer and allow for flexibility at its terminal regions (Supplementary Fig. 7). The 30-nm extended region is composed of the hetero-tetrameric AUG1,3,4,5 assembly, while the V-junction is composed of AUG2,6,7,8 C-terminal regions assembled onto a platform composed of AUG3,5 folding back onto themselves (Fig. 3A, B and Supplementary Figs. 5, 7, and 8). The belly region contains AUG1,4, which binds the N- and C-terminal parts the central section of AUG3,5 (Fig. 3B). The belly region is composed of an α-helical bundle (Fig. 3B). The AUG1,3,4,5 C-termini form four helical bundles that supertwist together in the leg region and then fold their C-termini together into the tripod region (Figs. 2D and 3B). In the tripod, the AUG1,3,4,5 diverge into two subdomains where AUG1,4 fold into a larger spoke while the AUG3,5 C-termini form a second shorter spoke (Figs. 2D and 3B). The final and wider spoke in the tripod consists of the AUG3,5 N-terminal bundle, which extends from AUG3,5 N-terminal helices (Fig. 3B and Supplementary Fig. 5M). The tripod region was not predicted accurately by AlphaFold (Supplementary Fig. 8C). From the top end of belly region, the stem of the V-junction emerges from two sets of helices of the AUG3 and AUG5 with opposite topologies (Figs. 2E and 3B and Supplementary Fig. 8C). This region is a platform where AUG3,5 subunits fold back and assemble with AUG2,6,7,8 subunits to form the V-junction (Fig. 3B, top panels). In the AUG1,3,4,5 assembly, the stem is not ordered, likely due to the destabilization of the AUG3,5 foldback zone in the absence of AUG2,6,7,8 (Supplementary Figs. 7F and 8C). The AUG2,6,7,8 C-termini form four helical bundles that can be followed into the V-junction arm, including the AUG6,7 N-terminal dual CH domains (Fig. 3B). Our model differs from previous AlphaFold models in that the helical regions of AUG3,5 foldback zone undergoes more extensive folding with AUG6 and AUG2 C-termini (Figs. 2A, B and 3B and Supplementary Fig. 8A, B).

Augmin structure stabilized by long and short range coiled-coil interfaces
The structures reveal the full complex topology of all eight Augmin subunits (Fig. 3B). AUG3 and AUG5 form a heterodimeric parallel long coiled-coil that form the backbone of the Augmin structure (Fig. 3B, left panel). AUG1,4 subunits fold into the lower half of the extended region, and they stabilize their N and C-terminal regions by folding in parallel with AUG3,5 N-term cluster in the leg while anti-parallel folding into against themselves into the belly region with both N and C-terminal central helices of AUG3,5, giving this region its girth with an eight helical bundle (Fig. 3B, lower left). At the end of the extended region of Augmin, the tripod consists of a co-folded AUG1,4 C-terminal bundle as the longest spoke and AUG3,5 C-terminal bundle, which forms a second shorter spoke, and the AUG3,5 N-terminal cluster forming the third wider spoke of the tripod (Supplementary Fig. 5M and Fig. 3B, lower panel). In the V-junction of Augmin, the AUG2 and AUG6 C-termini intertwine within the AUG3,5 fold back, forming a six helical bundle leading the stem to widen just below the V-junction (Figs. 2A, B and 3B, top right). The AUG2,6,7,8 C-termini stabilize the AUG3,5 helices in the foldback zone at the top V-junction on while their N-terminal domains towards the long end of the V-junction to form the head globular region (Fig. 3B, top left). The two sets of N- and C-termini of AUG3,5 fold onto AUG1,4 fold back zone, leading to the belly region in the center (Fig. 3B, lower panel). The Augmin model reveals a mixture of parallel and anti-parallel intertwined coiled coils stabilized by short and long-range foldback interactions leading to its conserved organization (Supplementary Fig. 8B, D and Fig. 3B).

We resolved two states of the AUG6,7 CH-domains in the head region: an open and closed state of the AUG6,7 CH domains (Fig. 2B, left panel; and Supplementary Fig. 6G–J). In the closed state, the CH domains are aligned laterally tightly against each other (Fig. 2A, left panel and Supplementary Fig. 6H), and in the open state, they are splayed apart (Fig. 2A, right panel and Supplementary Fig. 6G). These transitions are coupled with a lateral rotation of the AUG2,6,7,8 helical bundle (Fig. 2B, right panel). Taken together, the Augmin V-junction undergoes transitions continuously stretched throughout the structure, where stabilization in one part would likely stabilizes subsequent parts (Supplementary Fig. 7). Due to flexibility in this region, we were unable to determine a high-resolution density map of the V-junction (Supplementary Fig. 7).

Four critical conserved surfaces can be identified on the Augmin structure
To understand the sequence conservation and charge distribution, we aligned sequences for each of the hetero-octameric subunits (AUG1,2,3,4,5,6,7,8) across two hundred orthologs from plants, animals, and insects. We plotted sequence conservation and compared those to a charge distribution plot along the surface of the Augmin model (Supplementary Fig. 9 and Fig. 3D, E). We observe extensive conservation in the coiled-coil helical interactions between subunits within various regions of the structure, suggesting conserved long and short-range assembly interactions (Supplementary Fig. 9A–C and Fig. 3E). Conservation on the surface of the Augmin hetero-octamer reveals four conserved regions with distinct charge distributions, likely indicating critical zones of functional Augmin protein interaction zones (Supplementary Fig. 9A–C and Fig. 3D, E). (1) a large surface on the backside of the belly region involving interfaces with AUG3,5 and AUG1,4 N-terminal helical regions; (2) the AUG3,5 C-termini, and the AUG1,4 C-termini in the tripod (Fig. 3D, E); (3) the top of V-junction interfaces composed of the AUG3,5 central foldback onto the AUG2,6,7,8 C-terminal helical bundle (Fig. 3D, E); this region of the V-junction is bound by the density which we assigned to the RplB β-barrel, which likely forms the binding site for NEDD1-β-propeller (see below); and 4) the AUG6,7 dual CH domain dimer in the head region, which mediates MT binding (Fig. 3D-E).

Augmin undergoes antiparallel dimerization via the tripod to belly regions
2D-class averages of full Augmin hetero-octameric (AUG1,2,3,4,5,6,7,8) particles show side views of a “head-to-tail” Augmin homodimers bound to each other along their long axis (Fig. 1E, fourth panel; Supplementary Fig. 2, left panels). 2D-class averages for the 1.5 Augmin particle show an identical “head-to-tail” dimer of extended regions but are missing a V-junction and stem regions from one of the two assemblies (Fig. 1E, third panel). We determined a 12-Å C2 symmetric anti-parallel cryo-EM single particle structure followed by extended region model placement and de novo model refinement (Supplementary Fig. 2, left panels; Fig. 4). In this structure, the V-junctions and part of the stem are 30 nm away from the center and were plagued with higher flexibility, and were thus excluded (Supplementary Fig. 2, left panels). Despite the 12 Å resolution of the central region structure, we were able to place the Augmin core models into each Augmin protomer in the head-tail dimer structure and understand the conformational transitions they undertake upon dimerization (Fig. 4B, C and Supplementary Fig. 10). Dimerization is mediated by one face of the belly region and the backside of a rearranged tripod region (Fig. 4B–D and Supplementary Fig. 10). In the Augmin dimer state, the tripod region undergoes a 20° rotational re-arrangement leading repositioning of its three lobes to be face outwards away from the dimerization site (Fig. 4E). The AUG3,5 N-terminal cluster lies close to the dimerization site on the back of the AUG1,4 C-terminal lobe and binds to the belly domain of the second Augmin particle, which is anti-parallelly oriented (Fig. 4D). The belly region and the leg region both twist in their folds, accommodating the rearrangement in the dimeric Augmin state. The surfaces of the tripod and the belly regions mediating the head-to-tail Augmin dimerization are highly conserved in the monomeric Augmin particle (Fig. 3C–E). The Augmin dimer structure reveals the conformational transitions in the extended region during anti-parallel dimerization.

Crosslinking mass spectrometry validates Augmin assembly interfaces
To understand the multi-subunit coiled-coil interactions within the Augmin hetero-octamers, we carried out crosslinking mass spectrometry (XL-MS) for both hetero-tetrameric (AUG1,3,4,5) and hetero-octameric (AUG1,2,3,4,5,6,7,8) assemblies (Fig. 5; Supplementary Fig. 11I; Supplementary Data, see “Methods”). Two sets of crosslinking analysis were performed, in which top contaminating proteins from bacteria were either excluded or included in the search database (Fig. 5 and Supplementary Figs. 11 and 12). Inclusion of bacterial proteins in the extended XL-MS analysis revealed crosslinks between the Augmin hetero-octamer and several copurifying bacterial proteins. This analysis was used as the basis for identifying RplB protein which binds to the V-junction of AUG1,2,3,4,5,6,7,8 (see above; Fig. 2). Overall, we consider the results from the more restricted search database to be more authoritative, as they yielded more identifications and because the crosslinker used (BS3) is non-cleavable, making it less suited for searches against larger databases (cf. Supplementary Data 1). Therefore, the results presented below are based on the restricted search.

Plotting the Cα-Cα distances for the 216 identified crosslinks that can be fit within the Augmin hetero-tetramer (AUG1,3,4,5) cryo-EM model reveals that 122 (56%) of these crosslinks fall below a 35 Å cut-off, and are shown in Fig. 5A (see also Supplementary Fig. 11A, D, G). When we interrogated where in the structure crosslinks occurred, we found many close-range inter-subunit and intra-subunit crosslinks within the belly region, the foldback region, and the tripod assembly, supporting the assignment of the chains in the cryo-EM density (Fig. 5A). Notably, we detected nine crosslinks that connect residues far in primary sequence, linking the AUG3,5 C-termini to the AUG3,5 N-termini, and 41 crosslinks between AUG3 and AUG5 in the foldback zone (residues 225–378 in AUG3 and residues 342–495 in AUG5) supporting a key feature of the structural model that the AUG3 and AUG5 are elongated coiled-coils that originate in the tripod assembly proceed to fold back, and terminate back into the tripod assembly as well. Crosslinks whose Cα-Cα distances exceed the 35Å threshold involve the residues assigned to the tripod, suggesting that this region is more dynamic in the hetero-tetrameric (AUG1,3,4,5) assembly. The extensive crosslinks in the AUG3,5 central foldback zone that fall within the 35Å cutoff are notable, given that this region was not resolved in the AUG1,3,4,5 cryo-EM structure. The data suggest that this region may be too flexible to be resolved by cryo-EM in the absence of AUG2,6,7,8, but the overall structure of AUG3,5 is nevertheless supported by XL-MS (Fig. 2C and Supplementary Figs. 8C and 11D, E).

Plotting the Cα-Cα distances for the 115 identified crosslinks that can be fit within the Augmin hetero-octamer (AUG1,2,3,4,5,6,7,8) cryo-EM model reveals that 85 (74%) of these crosslinks fall below a 35 Å cut-off and are shown on the model in Fig. 5B (see also Supplementary Fig. 11B, E, H). That a higher percent of the crosslinks comport with the cryo-EM model, and suggest the octamer is a less dynamic assembly, which is consistent with our biochemical studies (Fig. 1 and Supplementary Fig. 1). We observed a few inter-subunit and intra-subunit crosslinks in the region forming the V-junction, where the AUG2,6 C-termini interact with the AUG3,5 foldback zone. The AUG2,6,7,8 crosslinks are observed in both the bow region and the AUG6,7 dual CH domains in interfaces with AUG2,8 (Fig. 5B and Supplementary Fig. 11E, H).

We measured the Cα-Cα distances between residue pairs in both the context of the monomeric Augmin assembly and the anti-parallel dimer model. Because the dimer model lacks the V-junction, only 59 of the 115 crosslinks could be mapped to the dimer model, but of those 59, 32 had even shorter Cα-Cα distances in the anti-parallel dimer model, suggesting that inclusion of the anti-parallel dimer state allows us to better capture some of the crosslinking data (Fig. 5C and Supplementary Fig. 11C, F). Of those 32 crosslinks, we identified 8 crosslinks in the tripod and leg regions spanning across dimers (Fig. 5C). The tripod and leg regions undergo conformational changes and become closer in the Augmin anti-parallel dimer model (Figs. 4 and 5C). Thus, these XL-MS data are consistent with our de novo Augmin model and support anti-parallel dimerization of the extended region via the conserved interfaces in the belly and tripod regions (Figs. 3E and 5B, C).

NEDD1 WD-β-propeller binds Augmin via AUG2,6,7,8 subunits
We next reconstituted the NEDD1 WD β-propeller with the hetero-tetrameric (AUG1,3,4,5) and hetero-octameric (AUG1,2,3,4,5,6,7,8) Augmin assemblies to define the location of the NEDD1 Augmin binding site (Fig. 6A, B). Full-length A. thaliana NEDD1 was insoluble in bacteria or insect cells. In contrast, the conserved N-terminal NEDD1-WD β-propeller (residues 1–315) is soluble when expressed in insect cells and purified as monodisperse protein (Fig. 6A, B and Supplementary Fig. 13A, B). The NEDD1-WD β-propeller was previously observed to bind MTs and synergize with Augmin20. We used sucrose density gradients to explore NEDD1 WD β-propeller binding to AUG1,3,4,5 and AUG1,2,3,4,5,6,7,8 (Fig. 6B and Supplementary Fig. 13C–H). The AUG1,2,3,4,5,6,7,8 bound NEDD1-WD β-propeller, and they both co-elute at high molecular weight with Augmin subunits, with excess NEDD1-WD eluting at lower molecular weight (Supplementary Fig. 13F–H). In contrast, NEDD1-WD β-propeller did not co-elute with AUG1,3,4,5 (Supplementary Fig. 13C–E). Thus, the NEDD1 β-propeller binding site is missing in the AUG1,3,4,5, but is present in AUG1,2,3,4,5,6,7,8 (Fig. 6B and Supplementary Fig. 13C–H). We validated the AUG1,2,3,4,5,6,7,8 interaction with NEDD1 WD-β-propeller using size exclusion chromatography (Fig. 6C, D). MP on sucrose density purified NEDD1-WD β-propeller AUG1,2,3,4,5,6,7,8 compared AUG1,2,3,4,5,6,7,8 to suggest similar mass (450 vs 460 KDa); however, NEDD1-WD β-propeller binding slightly induces Augmin dimerization (987 kDa), compared to its absence, where Augmins are present mostly as monomers in solution (Supplementary Fig. 13I, J). These data are consistent with location of the NEDD1-WD β-propeller binding site to be only present in AUG1,2,3,4,5,6,7,8, and is missing in AUG1,3,4,5, suggesting it lies in the V-junction, which is composed of AUG2,6,7,8 (Fig. 6A–D and Supplementary Fig. 13A–F). NEDD1 binding also mildly enhances the dimerization of Augmin particles (Supplementary Fig. 13I, J).

Cryo-EM structure of Augmin-NEDD1-WD β-propeller reveals its V-junction binding
To determine the NEDD1-WD-β-propeller binding site on Augmin, we collected cryo-EM data for complexes of AUG1,2,3,4,5,6,7,8-NEDD1-WD-β-propeller. Initial 2D class averages show a 50% bigger mass inside the V-junction where we the unknown barrel-shaped density binds Augmin. These 2D classes also show increased proportions of Augmin dimers and no 1.5 Augmin classes. We then determined a 10-Å cryo-EM structure revealing a larger mass, with circular shape and central hole, attached to the base of the V-junction (Fig. 6E–G). This density is flipped by 90° and has a wider and circular “donut” like shape. Although this map is of lower resolution, we observe conformational change in the V-junction region, tilting upwards, upon placement and morphing of our previously determined Augmin model. We were able to place the AlphaFold predicted NEDD1-WD β-propeller model into the additional density inside the V-junction, revealing that the NEDD1 β-propeller binds via its narrow side interface orientation inside the Augmin V-junction (Fig. 6F, H, I). We plotted the conservation of plant and animal NEDD1 orthologs on the fitted NEDD1 WD-β-propeller AlphaFold model (Fig. 6I). The conservation plot shows that the most conserved surface residues in NEDD1-WD β-propeller overlap extensively with the interface between the fitted NEDD1 and Augmin V-junction model (Fig. 6I). Furthermore, our low-resolution cryo-EM structure and resulting model for the NEDD1-β-propeller-Augmin V-junction is fully consistent with our biochemical reconstitution studies (Fig. 6B–D). The proximity of the NEDD1-WD β-propeller binding site at the base of the V-junction to AUG6,7 MT binding CH-domain dimer at the tip of the V-junction suggests that NEDD1-WD β-propeller MT binding may stabilize the conformation of Augmin’s V-junction to the MT lattice. We propose that the NEDD1-WD β-propeller binds both Augmin and MTs. The Augmin AUG6,7 CH-domain dimer also binds to MTs. The dual binding of Augmin-NEDD1-WD β-propeller likely leads to extensive contact with multiple MT protofilaments (model described below; Fig. 8A).

Coevolutionary analysis supports Augmin organization and its NEDD1 binding site
We investigated the co-evolution of AUG1,2,3,4,5,6,7,8 subunits and NEDD1 across the eukaryotic kingdoms (Supplementary Fig. 15A) which revealed conservation of all the nine proteins across plants and metazoans, including chordates such as mammals and fish, amphibians, and a small subset of insect species (Fig. 7A and Supplementary Fig. 15A). A significant number of flowering plants were found to possess the complete set of Augmin subunits and NEDD1, highlighting their crucial role in MT nucleation within plants (Fig. 7A, B and Supplementary Fig. 15A, B). To explore the co-evolution of Augmin hetero-octameric subunits, we designed a workflow that computed coevolutionary couplings of residues obtained from multiple sequence alignments (MSA) of pairwise combinations of Augmin subunits (see “Methods”; Supplementary Fig. 15D). We evaluated twenty pair-wise combinations of the Augmin subunits (Supplementary Table 1) and implemented Direct coupling analysis (DCA) to evaluate each MSA of the subunit pairs. DCA was used to quantify and rank the direct couplings between residue pairs based on Direct Information (DI) hereafter denoted as DI pairs (Supplementary Fig. 15D). DI and related metrics have been shown to be a proxy for physical contacts in protein complexes32–34 Only statistically significant DI pairs (p-value ≤ 0.2) were retained, including 42 DI pairs (Table S2), which we displayed on the cryo-EM Augmin model (Fig. 7C) and display on linear AUG1,2,3,4,5,6,7,8 connectogram (Fig. 7D). Mapping the DI pairs onto the structural model of AUG1,2,3,4,5,6,7,8 reveals strong correspondence in subunit pairings across the Augmin V-junction, dual-CH domains, extended, the tripod regions (Fig. 7C).

We performed a coevolutionary analysis of NEDD1-WD β-propeller with Augmin subunits using the seed sequences from humans, plants and insects (see “Methods”, Supplementary Fig. 15C). A species occurrence heatmap was constructed, revealing the co-evolution of NEDD1 and the eight Augmin subunits across species from various taxonomic groups (Fig. 7B). The evolutionary analysis identified 461 species which contain all 9 proteins including 269 flowering plants, 180 chordates and 12 insects (Supplementary Tables 3 and 4 and Supplementary Fig. 15A). To independently identify the NEDD1-WD β-propeller binding site on the Augmin hetero-octameric assembly, DCA was carried out between NEDD1-WD β-propeller and AUG2,6,7,8 subunits (see “Methods”). The identified DI pairs between NEDD1-WD β-propeller and Augmin subunits were then used to guide a molecular dynamics simulation, which brought them together at the V-junction of the Augmin hetero-octamer, revealing a model that is very similar to the cryo-EM structure with NEDD1-WD β-propeller binding to the AUG2,6,7,8 V-junction (Supplementary Table 5 and Figs. 7E and 6). These findings offer an independent line of evidence supporting the biological relevance of the functional interfaces between Augmin hetero-octamer and the NEDD1-WD β-propeller.

Methods
Cloning, protein expression, and purification
A. thaliana (At) Augmin AUG1,2,3,4,5,6,7,8 subunits and other plant orthologs were aligned with metazoan and insect counterparts, revealing high conservation The C-terminal region (residues 338–644) of AUG8 contains a highly conserved α-helical domain, while its divergent, disordered N-terminal domain containing the MT binding region was excluded.

AUG2,6,7,8 sequences contained multiple plant-specific rare Arg codons absent in bacteria, necessitating codon optimization. Each AUG1,2,3,4,5,6,7,8 subunit ORF was cloned into pET3a vectors and assembled into polycistronic co-expression constructs (Fig. 1A and Supplementary Fig. 1F) with C-terminal 8X his tags on AUG5 (His) and AUG6-StrepII tag (Strep) (Supplementary Fig. 1A). AUG1,3,4,5 subunits were assembled into a polycistronic vector with AUG5 C-terminal GFP and His tags (Supplementary Fig. 1F).

The At NEDD1-WD β-propeller (residues 1–315) domain was cloned into pFastBac with strepII tag. The Augmin Polycistronic constructs were transformed into SoluBL21 (AMSBio) cells on ampicillin plates. Hetero-octameric (AUG,1,2,3,4,5,6,7,8) Augmin was overexpressed from overnight cultures diluted 1:200 into 2xYT media with 200 µg/mL ampicillin. Cells were induced with 0.5 mM IPTG at OD 0.6, grown at 18°C for 12–14h, and harvested by centrifugation at 3000 rpm for 25 min. Pellets were resuspended in lysis buffer (50 mM HEPES pH 7.5, 200 mM KCl, 1 mM MgCl, 1 mM EGTA, 12 mM ß-mercaptoethanol, 10% (v/v) glycerol) with protease inhibitors (5 µg/mL leupeptin, pepstatin aprotinin, 0.1 mM PMSF, 5 µg/mL benzamidine, EDTA-free mini tablets (Sigma Aldrich). DNase I was added and cells lysed by microfluidization. Clarified lysates (18,000 rpm, 20 min, 4°C) were loaded onto recycled Ni-IDA columns (Macherey Nagel). Augmin was eluted with 200 mM Imidazole after washing. Assembly was verified by SDS-PAGE. Eluates were loaded onto Hitrap-QFF columns (Cytiva), and flow-through was further purified on Superose 6 16/60 columns in 50 mM HEPES pH 7.5, 200 mM KCl, 1 mM EGTA, 1 mM MgCL2, 10% (v/v) glycerol. The hetero-tetrameric (AUG1,3,4,5) Augmin was purified similarly but eluted from Q-FF with ~400 mM KCl and gel filtered on Superdex 200 16/60 (Cytiva) in 50 mM HEPES pH 7.5, 300 mM KCl, 1 mM MgCl2, 1 mM EGTA, 5% (v/v) glycerol. Aliquots (3 mg/mL) supplemented with 15% (v/v) glycerol were snap-frozen.

At NEDD1-WD β-propeller expressed in Tni cells (Expression Systems, Davis CA, catalog number 94-002S) (1:20 virus inoculum) 27°C for 60–70h. Cells (≥90% viability) were lysed in 50 mM HEPES pH 7.5, 250 mM KCl, 1 mM MgCl2, 1 mM EGTA, 5% (v/v) glycerol with protease inhibitors. NEDD1-WD was purified on regenerated Strep-Tactin columns (IBA) with 100 mM D-biotin elution and gel filtered on Superdex 200 16/60 (Supplementary Fig. 12) in lysis buffer. Aliquots (1 mg/mL) with 20% (v/v) glycerol were snap-frozen.

Purified full-length Augmin subunits were analyzed by LC-MS/MS (Taplin Facility). Gel bands were excised from 12% SDS-PAGE, digested, and identified peptides manually mapped.

Mass photometry and multi-angle light scattering experiments
MP experiments were performed using a Refyen 1.0 mass photometer. Data were analyzed in Discover MP software using mass calibration provided by Refyen. Samples (Supplementary Figs. 1D, H and 12G, H) were crosslinked with a ~50–100-fold molar excess of glutaraldehyde, then diluted to 5–10 ng/mL in 12 µL 20 mM HEPES pH 7.0, 100 mM KCl. Non-crosslinked samples were analyzed without dilution in 50 mM HEPES pH 7.5, 150–300 mM KCl, 1 mM MgCl2, 1 mM EGTA.

SEC-MALS was performed using an HPLC system equipped with a Superdex 200 Increase 10/300 column in line with a Wyatt Mini-DAWN TREOS multi-angle light scattering detector and Optilab reflective index detector (Wyatt Technologies). AUG1,2,3,4,5,6,7,8 (200 µL, 1 mg/mL; Supplementary Fig. 1B) or AUG1,3,4,5 (200 µL, 1 mg/mL; Supplementary Fig. 1G) were injected and eluted in 50 mM HEPES pH 7.5, 200 mM KCl, 1 mM MgCl2, 1 mM EGTA. Molecular masses were determine using ASTRA software (Wyatt Technologies).

Augmin and NEDD1-WD β-propeller binding experiments
Reconstitutions of hetero-octameric (AUG1,2,3,4,5,6,7,8) with NEDD1 was evaluated was by size exclusion chromatography on a Superose 6 5/150 column (Cytiva) in 50 mM HEPES pH 7.5, 150 mM KCl, 1 mM MgCl2, 1 mM EGTA, 5% (v/v) glycerol with 100 µL injections. AUG1,2,3,4,5,6,7,8 at 1 mg/mL was incubated with and without a ten-fold molar excess of NEDD1-WD. Sucrose density gradient experiments were performed with AUG1,2,3,4,5,6,7,8 (~1 mg/mL) or AUG1,3,4,5 (~2 mg/mL) in the presence or absence of a five fold molar excess of NEDD1-WD on 10–40% sucrose gradients in 50 mM HEPES pH 7.5, 150 mM KCl, 1 mM EGTA, 1 mM MgCl2 and fractions were evaluated using SDS PAGE (Supplementary Fig. 12C–H)

Cryo-EM sample preparation and data collection
The hetero-octameric (AUG1,2,3,4,5,6,7,8) and Hetero-tetrameric (AUG1,3,4,5) Augmin samples were purified by size exclusion chromatography on Superdex 200 10/300 columns (Cytiva), concentrated to 1 mg/mL, and crosslinked with 200 nM BS3 (Thermo Fisher) on ice for 2h, followed by quenching with 10 µM Tris-HCl pH 8. AUG1,2,3,4,5,6,7,8-NEDD1-WD-β-propeller complexes were purified on a Superdex 200 16/60 column, concentrated to 3 mg/mL, crosslinked with 1 µM BS3 for 1h, and quenched with 1 mM Tris-HCl pH 7.0. Buffers are described in the Supplementary Information.

Cryo-EM grids were prepared using a Leica EM GP2 with sensor blotting at 20°C and 95% humidity, 5–10 s pre-blot, 5–8 s blot time, and 1.5–1.8 mm extra push. R1.2/1.3 300 mesh grids (Quantifoil) were used (Cu for AUG1,2,3,4,5,6,7,8 and AUG1,3,4,5; Au for AUG1,2,3,4,5,6,7,8-NEDD1-WD). Some AUG1,2,3,4,5,6,7,8 grids included 0.001% NP-40.

Grids were screened on a Glacios microscope (Thermo Fisher) with a K3 direct electron detector (Gatan) at 200 kV using SerialEM38 at 11,000× and 45,000× magnifications using low dose conditions. High-resolution data were collected at 45,000× (0.44 Å/pixel) in Super-resolution mode) with SerialEM38 and beam-image shift39, at −0.6 to −1.8 µm defocus and 60 e−/Å2 total dose over 75 frames.

Cryo-EM single particle image processing
The full Augmin hetero-octamer (AUG1,2,3,4,5,6,7,8) structure (Supplementary Fig. 2, black arrows) was determined as follows: 5717 movies of hetero-octameric (AUG1,2,3,4,5,6,7,8) Augmin were motion-corrected (MotionCor240, 2× binned) and CTF-estimated (CTFfind41) in RELION42. Particles (20 million) were picked by LoG-based template-free picking (50–450 Å diameter) and extracted (200 pixels, 3.52 Å/pixel). After 2D classification in cryoSPARC43, 714,446 particles were re-extracted in RELION42 (300 pixels, 1.76 Å/pixel). Further 2D- and 3D-classification. Using ab initio model generation followed by refinement yielded a 9.6 Å map from 180,160 particles, 3DFlex44 refinement in cryoSPARC43 followed by 2D-classification and homogeneous refinement produced a 9.1 Å map based on Fourier shell correlation (FSC). Local resolution was calculated in PHENIX45.

The Augmin V-junction-stem structure (closed state) structure (Supplementary Fig. 3) was determined as follows: Particles were re-centered on the V-junction and re-extracted (160 pixels, 1.76 Å/pixel). 2D-classification selected 440,976 particles, which were 3D-refined, CTF-refined, and polished46 in RELION42. Duplicate-removed particles (223,355) were re-extracted (200 pixels, 1.76 Å/pixel), and ab initio model was generated and refined in cryoSPARC43. Heterogeneous refinement with three ab initio models yielded 107,694 particles in the closed state. Further 2D-classification and 3DFlex44 refinement, followed by re-extraction, 2D- and 3D-refinement, produced a 7.3 Å closed V-junction map from 75,828 particles. A second dataset of hetero-octameric (AUG1,2,3,4,5,6,7,8) Augmin 11,256 movies was processed similarly, and particles from both datasets (200,853) were merged in cryoSPARC43. 2D, heterogeneous, non-uniform47, and local refinement, combined with 3D variability analysis48 and re-extraction (256 pixels, 1.76 Å/pixel), yielded a final 7.3 Å map from 18,243 particles (3DFlex44 refinement, DeepEMhancer49 sharpening).

The Augmin V-junction-stem (open state) structure (Supplementary Fig. 3, grey arrows) was determined as follows: 94,659 particles from heterogeneous refinement of the closed state were 3D refined, 2D classified, and re-extracted (200 pixels, 1.76 Å/pixel) to select 67,132 particles. Homogeneous, non-uniform47, and 3DFlex44 refinement, followed by 3D variability analysis48 and individual frame reconstruction, selected 37,048 particles, 3DFlex44 refinement, CTF refinement, and non-uniform refinement produced a 12 Å open V-junction map, after sharpened using DeepEMhancer49.

The Antiparallel Augmin dimer structure (Supplementary Fig. 2, blue arrows) was determined as follows: 6578 particles from 2D classification were used to generate an initial dimer map in cryoSPARC43, which was refined in RELION42. Particles were re-centered on the AUG1,3,4,5 overlap region and re-extracted (384 pixels, 0.88 Å/pixel). Homogeneous and non-uniform refinement with C2 symmetry, followed by 3DFlex44 refinement, yielded an 8.1 Å map (B-factor sharpening).

The Augmin 1.5-mer structure (Supplementary Fig. 2, gray arrows) was determined as follows: 81,112 particles showing a second leg were separated by heterogeneous refinement, re-extracted (350 pixels, 1.76 Å/pixel), and 2D classified to select 11,903 particles. Ab initio reconstruction followed by homogeneous and non-uniform refinement produced a 12.4 Å 1.5-mer map.

The hetero-tetrameric (AUG1,3,4,5) Augmin extended region structure (Supplementary Fig. 4) was determined as follows: 10,830 movies were motion-corrected (MotionCor40, 2× binned) and CTF-estimated (GCTF50). 15.5 million particles were picked by reference-free LoG picking (80–350 Å diameter) and extracted (100 pixels, 3.52 Å/pixel). 2D classification in cryoSPARC43 selected 638,316 particles, which were re-extracted (200 pixels, 1.76 Å/pixel) and 3D refined in RELION42, followed by CTF refinement and polishing of 387,892 particles. Iterative 3D refinement, 2D/3D classification, and re-extraction (500 pixel, 0.88 Å/pixel) yielded a 4.1 Å map from 247,267 particles. 3D classification selected 144,287 particles, which were re-extracted (512 pixels, 0.88 Å/pixel) and refined in RELION42 and cryoSPARC43 to produce a 3.9 Å map. Re-extraction (400 pixels, 0.88 Å/pixel), 2D classification, and iterative refinement in cryoSPARC43 yielded a 3.5 Å map from 72,002 particles (3D variability analysis48, 3DFlex44 refinement, DeepEMhancer49 sharpening). Focused local refinement of the tripod with a tight mask produced a 5.9 Å map.

The Augmin V-junction-NEDD1-WD-β-propeller structure (Supplementary Fig. 14) was determined as follows: 8813 movies were motion-corrected (motioncorr2, 2× binned), CTF-estimated (CTFFind3), and blob-picked (50–600 Å diameter) in cryoSPARC43. 2D classification of 10 million initial particles (200 pixels, 3.52 Å/pixel) selected 15,844 particles used for template picking, yielding 510,466 particles. 2D classification selected 39,064 particles, which were used to generate an ab initio model and 3D refined. Particles were re-extracted (250 pixels, 1.76 Å/pixel), and 2D classification selected 27,025 particles. Template-picking along with 3D refinements, classifications, re-extraction, and local refinement with 49000 particles (180 pixels, 1.76 Å/pixel) to solve 10 Å (DeepEMhancer49 sharpening) map for Augmin (V-junction) with NEDD1-WD40.

Model building and refinement
Model interpretation was limited to lower than reported resolutions due to resolution estimate inflation likely caused by map flexibility and low signal-to-noise of elongated particles, a trend observed in all published Augmin reconstructions29–31. Interpretation was based on visual map features rather than nominal resolutions. The extended region (AUG1,3,4,5) maps ranging from 3.5 to 4 Å were interpreted to 4 Å. The AUG1,3,4,5 tripod density and closed V-junction CH-domain maps at 5.9 Å were interpreted to 7.3 Å for the full Augmin model. Augmin (V-junction) with WD40 was built using closed CH-domain model with placement of WD40 domain (AlphaFold). Open CH-domain, 1.5-mer, and dimer maps were interpreted at 12, 15, and 10 Å, respectively.

Initial models were built by rigid body fitting AlphaFold3 predictions (Supplementary Fig. 8) for AUG1,3,4,5 and AUG1,2,3,4,5,6,7,8 into density using UCSF ChimeraX51 and Coot52. The AUG3,5 fold-back zone and AUG2,6,7,8 regions were built into the 7.3 Å V-junction map by flexible fitting and manual adjustment based on AlphaFold3 secondary structure predictions. The open CH-domain conformation (Supplementary Fig. 6C) was modeled by rigid-body fitting into distinct CH-domain-likedensities.

The 3.7 Å AUG1,3,4,5 map was used for de novo modeling of the interacting helical regions in the belly and legs, starting from the AlphaFold prediction. AUG3,5 N-terminal and C-terminal helical bundles and AUG1,4 C-termini were built into the 6 Å map using secondary structure element length, connectivity, and interactions as guides.

A full de novo AUG1,2,3,4,5,6,7,8 model was assembled by merging the above V-junction-stem and extended region models and fitting into the 8.1 Å consensus map using a 20 Å, 4-helix bundle in the central AUG3,5 (Supplementary Fig. 5A, B). The dimer model was built by rigid body fitting two copies of AUG1,2,3,4,5,6,7,8 into the dimer map. The AUG1,2,3,4,5,6,7,8-NEDD1-WD model (Supplementary Fig. 14B) was built by fitting the closed V-junction model and placing NEDD1-WD into the junction density. All models were subjected to real space refinement in PHENIX45 (Table 1).

Crosslinking mass spectrometry (XL-MS) of Augmin assemblies
AUG1,2,3,4,5,6,7,8 and AUG1,3,4,5 samples were crosslinked with 0.5–2 mM BS3 at 4°C overnight, denatured (8M urea), reduced (5 mM DTT), alkylated (15 mM IAA), quenched with DTT, digested with LysC (1:100 w:w ratio), diluted fourfold, digested with trypsin (1:50 w:w ratio), desalted (Sep-Pak C18), and vacuum-dried. Desalted peptides were fractionated via Superdex 30 10/300 GL gel filtration, then dried and stored at −80°C. AUG1,3,4,5 fractions were analyzed using an UltiMate 3000 UHPLC system coupled to a Q-Exactive HF-X Orbitrap. AUG1,2,3,4,5,6,7,8 fractions were analyzed with a Vanquish Neo UHPLC coupled to Orbitrap Ascend. Peptides were loaded onto PepMap 100 C18 column with Solvent A (0.1% formic acid in water) and Solvent B (0.1% formic acid in ACN). AUG1,3,4,5 peptides were separated using PepMap RSLC C18 column with a linear gradient from 5% to 25% solvent B over 100 min, then to 45% over 25 min, and then to 90% over 5 min. AUG1,2,3,4,5,6,7,8 peptides were separated with the same column with a linear gradient from 10.4% to 32.8% solvent B over 87 min, then to 44% over 5.5 min, and then to 76% over 2.5 min. For LC-MS/MS data acquisition, the Q-Exactive HF-X performed MS1 scans at 120,000 resolution (350–1500 m/z), AGC target of 3×106, and 50 ms max IT. The top 10 precursors (z = 3–8) were isolated (1.4 m/z window) and fragmented using stepped NCE (30±6). MS2 scans were at 60,000 resolution (200–2000 m/z), AGC target of 8×103, and 150 ms max IT. Dynamic exclusion was set to 45s and in-source CID at 10 eV. For the Orbitrap Ascend, MS1 scans were at 240,000 resolution (380–2000 m/z), normalized AGC target of 150%, and 100 ms max IT. The top 20 precursors (z = 3–7 with 4–7 preferred) were isolated (1.4 m/z window) and fragmented (NCE 30±6). MS2 scans were at 60,000 resolution (150–2000 m/z), normalized AGC target of 750%, and 250 ms max IT. Dynamic exclusion was set to 30s and in-source CID at 10 eV.

RAW files were converted and recalibrated using the xiSEARCH preprocessing pipeline Python script. The recalibrated MGF files were then searched with xiSEARCH 1.8.6 for crosslinked peptides. Search parameters were used as following: MS1 mass tolerance, 6 ppm; MS2 mass tolerance, 10 ppm; allowed maximum number of missed cleavages, 4; minimum peptide length, 6. Crosslinks were searched based on the modifications at Lys and N-term (preferred), modifications at Ser, Thr, and Tyr were also allowed with lower priority. Carbamidomethylations (+57.021464 Da) on Cys were enforced as fixed modifications, oxidations (+15.99491463 Da) on Met were allowed as variable modifications. FASTA files containing AUG1,3,4,5 or AUG1,2,3,4,5,6,7,8 were applied in the search separately. Search results were filtered in xiFDR 2.3.2 at the residue pair level to an FDR of 5%. The boost function was enabled between residue pairs, and the rest of the settings were default. The mzid file generated from xiFDR was uploaded onto xiVIEW and PDB files generated from cryo-EM study were imported for crosslink visualization. The same RAW files for AUG1,3,4,5 or AUG1,2,3,4,5,6,7,8 were also analyzed using FragPipe 22.0 to search for linear peptides. The proteins with the top 50 most peptide-spectral matches were used to generate alternative FASTA files for expanded crosslink searches that include contaminant proteins. The same xiSEARCH/xiFDR pipeline was applied to the expanded FASTA database search, and the respective mzid files were uploaded onto xiVIEW without importing the PDB files. Figure 5 and Supplementary Fig. 11D–F were generated by exporting the filtered crosslinks from xiVIEW and drawn in ChimeraX51 with XMAS53. Connectograms in Supplementary Fig. 11G, H were generated with xiVIEW. Protein-protein interaction networks of AUG1,3,4,5 or AUG1,2,3,4,5,6,7,8 and contaminant proteins in Supplementary Fig. 12 were generated by xiVIEW.

Mapping coevolutionary interactionsthrough direct coupling analysis
Seed sequences and Hidden Markov model (HMM) profiles were obtained for the eight Augmin subunits from UniProt and Pfam (InterPro), respectively54. The HMM files containing the protein domains were used to search the UniProt database and generate MSA Fasta files, except for AUG1 for which there was no Pfam domain, and the complete seed sequence was used to create a custom made HMM to generate an MSA (Supplementary Table 3). Each MSA consisted of homologous protein sequences belonging to the same protein domain family from diverse species. Sequences with large gaps in MSAs (maximum continuous gap of 20% the length of sequence) were removed and the filtered MSAs were horizontally concatenated using the Progressive Paralog Matching algorithm (PPM)55,56. DCA was carried out on the paired MSAs to obtain the residue pairs across proteins with the largest coevolutionary coupling signals (Supplementary Table 1)55. To estimate the significance of classifying a DI pair as interacting, a hypergeometric test was performed and only the DI pairs with a p-value ≤ 0.2 were retained (Supplementary Table 3), which were mapped onto the Augmin structure using Chimera (Supplementary Table 2). The p-values in Supplementary Table 3 were calculated using right tailed Hypergeometric test, testing for enrichment of true positive inter-domain DCA contacts among a given number of predicted DI pairs.

NEDD1 coevolution and phylogenetic tree construction
Seed sequences for each of the eight Augmin subunits and NEDD1 were selected from Homo sapiens, A. thaliana, and D. melanogaster from UniProt. Each seed sequence was queried against the UniProt database using BLAST, and the top three hits per species, based on highest sequence identity and lowest e-value, were selected. This produced a total of nine representative sequences for each protein. These were aligned using Clustal Omega57 and HMM profiles were constructed from the resulting alignments. The HMM profiles were used to search the UniProt database and generate MSAs for each of the nine proteins. The MSA of full NEDD1 sequences was filtered to remove sequences with maximum continuous gaps of 50% caused due to insertions and deletions. Using NCBI Taxa from ETE358, species were categorized from each protein MSA into taxonomic groups, which are represented in Fig. 7B. Subsequently, species that were present in all nine MSAs were identified, and a phylogenetic tree of these common species was constructed using the NCBI Taxonomy Browser and iTOL59 presented in Supplementary Fig. 15A.

Computational modeling of interaction between NEDD1 and Augmin
To investigate the potential interface between NEDD1 and the Augmin complex, seed sequences corresponding to NEDD1-WD β-propeller and the V-junction regions of AUG2,3,5,6,7,8 were taken from the V-junction of the Augmin hetero-octamer sequence regions. These sequences were used to generate MSAs for each subunit, which were then horizontally concatenated in six NEDD1-AUG combinations using PPM (Supplementary Table 4). DCA was performed on each paired MSA to identify coevolving residue pairs with high DI score (Supplementary Table 5). To model NEDD1 docking onto Augmin, the partial structure file from 9NBI containing only the V-junction region was modified in Chimera such that NEDD1-β-propeller was positioned approximately 60 Å away from the core of the Augmin assembly. From this, a topology and a .gro (Gromos87) file were prepared using the SMOG server60.

The DI pairs from each NEDD1-AUG combination were combined and then ranked based on DI score and the DI pairs with a combined solvent accessible surface area of greater than 100 were retained61; the topology file was modified to include only the top 10 of these DI pairs under “pairs” and “exclusions” sections. Subsequently, coarse-grained molecular dynamics simulations were performed using GROMACS (version 4.5.4_sbm1.0) through a hybrid SBM+DCA (Structure-Based Modeling+Direct Coupling Analysis) framework. This approach used evolutionary constraints i.e., 5 out of the top 10 DI-ranked residue pairs (Supplementary Table 5) identified via DCA, to gradually guide NEDD1 toward the Augmin complex during the simulation, leading to a model presented in Fig. 7E.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The nucleation of microtubule branching mediated by Augmin is essential for the construction of the mitotic spindle and\ncortical microtubules in plant cells. In this study, the authors elucidate the structural mechanism of the Augmin complex in\nArabidopsis thaliana using Cryo-EM, cross-linking mass spectrometry, and biochemical analyses. The key findings are as\nfollows:\n1. The first high-resolution structural model of the plant Augmin complex, which differs from its counterparts in metazoans.\n2. Demonstration of anti-parallel dimerization through conserved interaction sites\n3. The dual CH domains located at the V-junction display either open or closed conformations, suggesting a mechanism for\nregulating binding to the microtubule lattice.\n4. The NEDD1 WD40 domain binds directly to the V-junction of Augmin, supporting its dimerization and enhancing its\ninteraction with microtubules.\nThe authors also suggest that NEDD1 and the CH domains of Augmin create a platform for the recruitment of γ-TuRC\nthrough cooperative binding.\nThis study represents a significant advancement in understanding the role of the Augmin complex in microtubule nucleation.\nThe insights derived regarding its structure are valuable and contribute significantly to the field. It is recommended that this\nmanuscript be considered for publication in Nature Communications, provided that the following concerns are addressed:\nMajor points:\n1. Validate the structural models using mutagenesis analyses, especially for the Augmin dimer and the Augmin/NEDD1-\nWD40 complex. Additionally, it is preferable to evaluate these mutants in vivo for comprehensive results.\nMinor points:\n1. Line 79: The referenced study appears to indicate that augmin recruits NEDD1 to microtubule, rather than the reverse.\n2. A recent study by Gao et al. (Nat Commun, 2025) suggests that the region corresponding to the tripod of plant Augmin\ninteracts with the NEDD1/GCP3/MZT1 module. It raises the question of whether the tripod region of plant Augmin can be\nintegrated into the proposed NEDD1/GCP3/MZT1/Augmin model as predicted by AlphaFold.\n3. The direct interaction between the NEDD1 WD40 domain and the V-junction of Augmin is noteworthy, particularly\nconsidering that human Augmin does not exhibit binding to the NEDD1 WD40 domain in pulldown assays, as reported by\nZhang et al. (J Cell Biol, 2022). Could the authors provide further insight into a potential explanation for this discrepancy?", "reviewer_2": "By examining in vitro reconstituted Arabidopsis Augmin complex using cryoEM and crosslinking mass spectrometry,\nAshaduzzaman et al. proposed a model elucidating how the cooperativity between Augmin complex and NEDD1 may\ngenerate a platform to recruit gamma-tubulin ring complex and activate branched microtubule nucleation. Overall, the\nexperiments are well-designed, and the data interpretation is appropriate. This work is suitable for publication in Nature\nCommunications after addressing some concerns.\nMajor concerns\n1. It is not convincing that the high electron density spot at the V-junction is potentially due to the association with nucleic\nacid. Can the authors provide some supporting evidence? The proposed beta-barrel from bacteria should not contribute to\nthe strong electron density. In particular, it is puzzling why this strong signal in 2D class averages does not lead to a well-\ndefined density in the 3D reconstruction.\n2. Line 205-207, if AUG2,6,7,8 move upward and AUG3,5 in the base also move upward, where will be the pivot point for the\nmovement?\n3. In the crosslinking mass spectrometry, the authors suggest the hetero-dimeric assembly is more dynamic than the hetero-\noctamer. However, I do not think this statement is well-supported by their data. It is particularly unclear why the authors use\nthe 30 Å cutoff.\n4. How does the co-expression of the NEDD1-WD40 beta-propeller change the distribution of 2D class averages?\n5. If a bacterial beta-barrel domain is copurified with the Augmin complex, the authors should be able to identify the unknown\nprotein binder in their crosslinking mass spectrometry. Can the author perform the proposed mass spectrometry analysis? If\nnot, what are the possible reasons?\nMinor concerns\n1. The section ‘cryo-EM imaging of Augmin reveal dynamic architecture and higher order oligomers’ is confusing. It might be\nhelpful to add a cartoon to show the referred structural domains.\n2. Figure 1Ev is not mentioned in the text.\n3. It will be informative to show the ratio of each 2D-class average shown in Figure 1E.\n4. Line 263. I think ‘opposite topologies’ is a confusing term.\n5. The section “Augmin hetero-octameric structure stabilized by long and short-range coiled-coil interactions” is repetitive.\nI suggest the authors revise these paragraphs.\n6. Figure 5 is very confusing with all the colors.", "reviewer_3": "In the paper entitled “Cryo-EM structures of the Plant Augmin reveal its intertwined coiled-coil assembly, antiparallel\ndimerization, and NEDD1 binding mechanisms,” the authors have determined cryo-EM structures that lead to a complete de\nnovo model of the coiled-coil assembly of the plant Augmin complex. They employed XL-MS approaches to validate these\nfindings. In their models, Augmin forms antiparallel dimers through conserved interfaces in its extended region. The authors\nalso describe how the WD40 β-propeller domains of NEDD1 directly bind to the Augmin V-junction, near the AUG6/7/8\nmicrotubule (MT) binding site.\nAltogether, this paper presents a model in which NEDD1 promotes Augmin’s MT binding and oligomerization through\ndownstream conformational effects, ultimately resulting in the recruitment of the γ-TuRC complex during MT branch\nformation.\nThe data presented are of high quality and of particular interest, especially given the cryo-EM analysis of such elongated\nparticles. The findings are well supported by clear figures and an appreciated supplementary video. Although the resolution\nachieved by cryo-EM was moderate in some regions (10–20 Å), the integration of multiple techniques (such as XL-MS and\ncomputational modeling) enabled the authors to propose a robust model where NEDD1 facilitates Augmin’s MT interactions.\nAs I am not an expert in cryo-EM, my primary focus was on the biochemical and cross-linking experiments. However, the\ncross-linking data are difficult to evaluate since the raw files are not available. Additionally, the exported search results do\nnot align with the manuscript, and thus a thorough revision of this section is necessary prior to publication.\nPoints to be addressed by the authors:\n• The authors state that they identified 209 cross-links (XLs) within the Augmin hetero-octamer. However, after extracting all\nidentified XLs from the provided .csv file, and removing duplicates and decoys, I arrive at a total of 191 unique XLs. Could\nthe authors clarify the origin of the reported number 209?\n• The two exported datasets differ significantly: one appears to be a raw export, including decoy and reverse database\nresults, while the other seems to be a pre-processed version. Could the authors please provide a detailed description of the\ndata processing pipeline and ensure consistent exports for both the tetrameric and octameric forms?\n• In Figure S11, the model contains significantly fewer XLs than the number of XLs reportedly identified, despite the caption\nstating: “Augmin model presented in ribbon format with the observed crosslinks generated in tube format.” The model clearly\ndoes not contain ~200 or even ~100 crosslinks. It appears that long-distance XLs (>30 Å) were excluded from the\nvisualization, as none are visible. If this is the case, this filtering criterion must be clearly stated in the figure caption.\n• The authors analyzed how many crosslinks from the hetero-octameric Augmin complex (AUG1–8) can be explained when\nallowing crosslinks to span across assemblies in the anti-parallel dimer model. While this is a valuable analysis, why is it\nlimited to the octameric assembly, when the tetrameric form (Figure S11E) also shows four homo-inter XLs, compared to\nonly two in the octameric form? This comparison should be expanded or explained.\n• Line 367: The statement that “many XLs are found, suggesting the region is folded but flexible,” is not necessarily accurate.\nA high number of XLs may also result from an unfolded region that is spatially close due to the folding and stabilization of\nother parts of the complex. This interpretation should be reconsidered or rephrased.\n• Line 382: The authors claim that “plots of our detailed XL-MS crosslinks on the de novo Augmin model are fully consistent\nwith the critical and distinguishing features of our structural model.” However, this seems overstated, given that many XLs\nexceed the expected BS3 crosslinking distance (typically around 35 Å, with some reported exceeding 100 Å). This\nstatement should be tempered and clarified, possibly acknowledging model limitations or alternative explanations.\n• Since “mass spectrometry also identified several contaminating β-barrel–containing proteins” that copurify with Augmin\nthrough multiple steps (as mentioned in the Supplementary list), these proteins should have been included in the database\nsearch used for the XL-MS analysis. Identifying binding sites between these β-barrel proteins and Augmin could have been\nhelpful for structural interpretation. If insufficient XLs were obtained from co-purified complexes, the authors might consider\nperforming a dedicated XL-MS experiment on the complex formed between the full Augmin hetero-octamer (AUG1–8) and\nthe NEDD1-WD40 β-propeller domain.\nMinor issues to be addressed:\nLine 129: Figure 1A does not illustrate the presence or absence of conserved α-helices; it only shows the construct length\nand the position of the tags.\nSimilarly, Line 139: Figure 1A does not provide any relevant information regarding the statement “Mass spectrometry\nconfirms each Augmin subunit is purified in the assembly and that the AUG6 C-terminal region is prone to degradation with\nmultiple polypeptide bands.” This should be clarified or corrected.\nLine 141: The manuscript references mass spectrometry data with “(Supplementary list file),” but the actual data are missing.\nPlease ensure that this file is included.\nLine 348: It is unclear which complex (tetramer or octamer) the reported values “50% (105 out of 209)” and “70% (80 out of\n115)” refer to. This should be explicitly stated."}, "author_response": "To Editor and Reviewers,\nWe would like to submit a revised version of our manuscript \"Cryo-EM structures of the Plant\nAugmin reveal its intertwined coiled-coil assembly, anCparallel dimerizaCon and NEDD1 binding\nmechanisms\" to Nature Communica.ons.\nWe would like to thank the reviewers for their helpful suggesCons. In the revised manuscript we\ninclude the following major revisions:\n1) We present a revised Crosslinking mass spectrometry (XLMS) analysis of bacterial\nprotein co-purifying with Augmin assemblies and idenCfy the RplB protein as the\npotenCal b-barrel protein binding to the Augmin V-juncCon. Using this informaCon we\nhave revised the presentaCon and discussion of the density bound to the Augmin V-\njuncCon. We have also uClized a variety of methods to try to produce more informed\ntheories about its potenCal contents.\n2) We have revised the presentaCon of the Crosslinking mass spectrometry (XL-MS) data to\naddress concerns of the reviewers and clarified much of the analyses and fully rewrote\nall secCons associated with XL-MS.\n3) We present a new coevoluConary analysis of Augmin subunits and NEDD1 across plants,\nchordates, insects revealing the high degree of conservaCon of each of the eight Augmin\nsubunits and NEDD1. We also present a direct coupling analysis (DCA) across all eight\nAugmin subunits to idenCty physical (direct informaCon) pairings of changed amino\nacids in relaConship to the structural model presented. The DCA analysis provides\ndetailed mulC-subunit distance interfaces that independently validate with the structural\nmodel of the Augmin assembly presented. The DCA analysis provides independent\nvalidaCon for the Augmin binding site for NEDD1 WD b-propellor being the V-juncCon\nregion.\n4) We present an improved cryo-EM single parCcle structure of the AUG1,2,3,4,5,6,7,8 -\nNEDD1 WD complex. This improved cryo-EM map shows improved density features of\nthe NEDD1-WD at this juncCon. We also carried out DCA analysis of the NEDD1 WD b-\npropellor to determine its interface with the Augmin V-juncCon composed of AUG3,5\nand AUG2,6,7,8. The DCA analysis provides further validaCon of the NEDD1-WD b-\npropellor binding site onto the Augmin V-juncCon in support of the biochemical and\nstructural analysis\nBelow is a point by point response to the reviewer comments\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe nucleaGon of microtubule branching mediated by Augmin is essenGal for the construcGon\nof the mitoGc spindle and corGcal microtubules in plant cells. In this study, the authors\nelucidate the structural mechanism of the Augmin complex in Arabidopsis thaliana using\nCryo-EM, cross-linking mass spectrometry, and biochemical analyses. The key findings are as\nfollows:\n1. The first high-resoluGon structural model of the plant Augmin complex, which differs from\nits counterparts in metazoans.\n2. DemonstraGon of anG-parallel dimerizaGon through conserved interacGon sites\n3. The dual CH domains located at the V-juncGon display either open or closed conformaGons,\nsuggesGng a mechanism for regulaGng binding to the microtubule laWce.\n4. The NEDD1 WD40 domain binds directly to the V-juncGon of Augmin, supporGng its\ndimerizaGon and enhancing its interacGon with microtubules.\nThe authors also suggest that NEDD1 and the CH domains of Augmin create a plaZorm for the\nrecruitment of γ-TuRC through cooperaGve binding.\nThis study represents a significant advancement in understanding the role of the Augmin\ncomplex in microtubule nucleaGon. The insights derived regarding its structure are valuable\nand contribute significantly to the field. It is recommended that this manuscript be considered\nfor publicaGon in Nature CommunicaGons, provided that the following concerns are\naddressed:\nWe thank the reviewer for their posiGve assessment about our work. We provide a detailed\npoint-by-point response to the reviewer’s revision suggesGons.\nMajor points:\n1. Validate the structural models using mutagenesis analyses, especially for the Augmin dimer\nand the Augmin/NEDD1-WD40 complex. AddiGonally, it is preferable to evaluate these\nmutants in vivo for comprehensive results.\nWe thank the reviewer for this suggesGon. Although this suggesGon is fair, because of the\nsubstanGal amount of work and cost associated with preparing, and isolaGng mutant augmin\nassemblies, the inclusion of these experiments would substanGally delay the publicaGon of\nthe current results. We think these studies would be more suitable for a separate study that\nfocuses on the structure in its biological context.\nMinor points:\n1. Line 79: The referenced study appears to indicate that augmin recruits NEDD1 to\nmicrotubule, rather than the reverse.\nThere are mulGple experiments presented in Zhang et al 2022 that show that NEDD1 and\nAugmin have their own independent interacGon with microtubules. We have revised the text\nto explicitly indicate that both Augmin and NEDD1 have their own interacGons with\nmicrotubules and their combined interacGon with each other in the complex presented in this\nmanuscript shows how they may form a more stable interface with microtubule laWce\nsuitable for anchoring the microtubule branch nucleaGon complex mediated by the gama-\ntubulin ring complex.\n2. A recent study by Gao et al. (Nat Commun, 2025) suggests that the region corresponding to\nthe tripod of plant Augmin interacts with the NEDD1/GCP3/MZT1 module. It raises the\nquesGon of whether the tripod region of plant Augmin can be integrated into the proposed\nNEDD1/GCP3/MZT1/Augmin model as predicted by AlphaFold.\nTwo recent studies have addressed the NEDD1- helical domain binding to the g-TURC (Gao et\nal 2025 and Hugo Muñoz-Hernández et al 2025). Both studies show that the NEDD1 C-\nterminus forms a four-helix bundle that binds the g-TURC. We have revised the text to add in\nthe potenGal binding of the AUG1,3,4,5 tripod to NEDD1/MTZ1/GCP3. However, the\nAlphaFold observaGon presented does not predict the accurate fold we describe here for the\nAUG1,3,4,5 foot in binding this assembly and thus the interface may be inaccurate as\npresented in those papers.\n3. The direct interacGon between the NEDD1 WD40 domain and the V-juncGon of Augmin is\nnoteworthy, parGcularly considering that human Augmin does not exhibit binding to the\nNEDD1 WD40 domain in pulldown assays, as reported by Zhang et al. (J Cell Biol, 2022). Could\nthe authors provide further insight into a potenGal explanaGon for this discrepancy?\nThe NEDD1 WD was shown to bind microtubules in the Zhang et al 2022 study. We believe it\nis possible that in the human system the affinity of the NEDD1-WD for Augmin is enhanced\nupon binding microtubules. It is possible microtubules play a crucial role in assembling the\nAugmin and NEDD1 complex. WE have revised the text to add this possibility.\nReviewer #2 (Remarks to the Author):\nBy examining in vitro reconsGtuted Arabidopsis Augmin complex using cryoEM and\ncrosslinking mass spectrometry, Ashaduzzaman et al. proposed a model elucidaGng how the\ncooperaGvity between Augmin complex and NEDD1 may generate a plaZorm to recruit\ngamma-tubulin ring complex and acGvate branched microtubule nucleaGon. Overall, the\nexperiments are well-designed, and the data interpretaGon is appropriate. This work is\nsuitable for publicaGon in Nature CommunicaGons amer addressing some concerns.\nMajor concerns\n1. It is not convincing that the high electron density spot at the V-juncGon is potenGally due to\nthe associaGon with nucleic acid. Can the authors provide some supporGng evidence? The\nproposed beta-barrel from bacteria should not contribute to the strong electron density. In\nparGcular, it is puzzling why this strong signal in 2D class averages does not lead to a well-\ndefined density in the 3D reconstrucGon.\nThe nature of the high electron density at V-juncCon has puzzled us too. We iniCally labeled its\nassociaCon with nucleic acid due to its 3-4-fold higher intensity than the protein material of the\nV-juncCon onto which it binds. Based on a reanalysis of our mass spec data collected of the\nisolated Augmin complexes with an expanded list of proteins in the search database, we now\nbelieve the density belongs to RplB, a ribosomal protein contaminant from E. coli that co-\nprecipitates with Augmin (see response to #5). It is possible that because E. coli proteins tend to\nbe quite rigid and stable (at least in comparison to Augmin which has an elongated and complex\nstructure), that its electron density is less diffuse leading to the observed difference in intensity.\nThe idenCfied protein is a ribosomal b-barrel protein with affinity to rRNA. If it co-elutes with\nrRNA, this could also explain why the density has 3-4-fold higher intensity than the augmin\nprotein subunits it is bound to. It is noteworthy, however, that this density lies at the binding\nsite for NEDD1-WD b-propellor, which is absent in the AUG1,2,3,4,5,6,7,8 expression system.\nHowever, reconsCtuCon of NEDD1 led to a more uniform electron density at the V-juncCon in\nthe AUG1,2,3,4,5,6,7,8-NEDD1 WD 2D-class averages and their Cryo-EM map (Figure 6 and\nFigure S13), suggesCng that NEDD1 likely can replace RplB in vitro.\n2. Line 205-207, if AUG2,6,7,8 move upward and AUG3,5 in the base also move upward,\nwhere will be the pivot point for the movement?\nWe believe Augmin has conCnuous coordinated movement and thus it is sparsely distributed\nacross throughout the structure of the V-juncCon as evident from our flexibility analysis. See\nFigure S7 which provides details into the flexibility of the V-juncCon.\n3. In the crosslinking mass spectrometry, the authors suggest the hetero-dimeric assembly is\nmore dynamic than the hetero-octamer. However, I do not think this statement is well-\nsupported by their data. It is parGcularly unclear why the authors use the 30 Å cutoff.\nThe XL-MS secCon has been rewrieen in the revised manuscript. The original reason for staCng\nthat the anC-parallel dimer was more dynamic was mostly moCvated by the observaCon that\nwe could create a full reconstrucCon for the hetero-octamer (containing the V-juncCon) but the\nanC-parallel dimer model only contains the extended region (and without density we can assign\nto AUG2,6,7,8). It is of course possible that this is due to various technical reasons and not due\nto the anC-parallel dimer being more dynamic, so we have removed statements suggesCng as\nmuch.\nThe 30 Å cut-off is based on the length of the spacer-arm in BS3. The fully extended BS3 has a\nmaximum arm of 11.4 Å. However, because lysine itself has a long side-chain and reacCons to\nthe crosslinker occur at the distal amine, the theoreCcal distance between alpha carbons on\ncrosslinked lysines is 24 Å if the chains are distended. However, due to the flexibility of proteins,\nvarious authors have considered Ca-Ca distances between 30 – 35 Å to be the range that would\nmake an idenCfied crosslink “consistent” with a given structural model.\nAs discussed in the text, the hetero-tetrameric assembly (AUG1,3,4,5) has 56% crosslinks with\nCα–Cα distance within 35 Å, whereas the hetero-octameric assembly has 74% crosslinks fell\nwithin this range. On the other hand, in the hetero-tetrameric assembly, we found 42 “long”\ncrosslinks (> 100 Å, 19%) connecCng distal regions (tripod, belly and stem), whereas there were\nonly 16 long crosslinks found in the hetero-octameric assembly. Hence, this consCtutes\nevidence that the hetero-tetramer is more dynamic, which is also consistent with the cryo-EM\nitself. So this is an argument that we now emphasize more in the revised manuscript.\n4. How does the co-expression of the NEDD1-WD40 beta-propeller change the distribuGon of\n2D class averages?\nThe NEDD1-WD b-propellor protein was not soluble in the same expression system as\nAUG1,2,3,4,5,6,7,8. Thus, we had to reconsCtute AUG1,2,3,4,5,6,7,8 (full Augmin) and NEDD1-\nWD b-propellor in vitro. In 2D class averages, we see a larger mass with more uniform signal for\nAUG1,2,3,4,5,6,7,8-NEDD1 WD b-propeller complex, compared to higher intensity smaller spot\nin the AUG1,2,3,4,5,6,7,8 alone. Also, we observed a larger proporCon of Augmin anC-parallel\ndimer formaCon than Augmin alone which is experimentally supported by our mass photometry\nresults. We have revised the text to include these details.\n5. If a bacterial beta-barrel domain is copurified with the Augmin complex, the authors should\nbe able to idenGfy the unknown protein binder in their crosslinking mass spectrometry. Can\nthe author perform the proposed mass spectrometry analysis? If not, what are the possible\nreasons?\nThank the reviewer for the suggesCon. To begin, we reanalyzed the fracCons (that we gathered\nwhen we were fracConaCng Augmin pepCdes) for linear pepCdes against the whole E. coli\nproteome, and idenCfied many E. coli protein contaminants comingled with Augmin hetero-\noctamer. Proteins with very high spectral counts include chaperones GroEL and DnaK, DeaD,\nand Pnp, but also many RNAP and Ribosome subunits. Although suggesCve that these proteins\ninteract with Augmin, co-precipitaCon can occur for many reasons, including non-specific\nbinding to resin. Using these results however, we re-performed a crosslinking search on the\nsame mass spectra with a limited search database containing the top 50 proteins (42\ncontaminants for hetero-octamer and 46 contaminants for hetero-tetramer). The results show\nindeed that several E. coli proteins bind to Augmin, including Pnp, DnaK, and several ribosomal\nproteins (RpmA, RplB, RplD, RpsD). The XiView visualizaCon of this map is shown in the paper\nas Figure S12. Notably, RplB’s crosslinks target it to the V-juncCon, which is evidenced by two\ncrosslinks, to K401 and K747 on AUG5 (which are both located at the V-juncCon of the Augmin\ncomplex). Hence, we tentaCvely assign this density to RplB (see above comment regarding\nunknown V-juncCon density). We do think that the evidence we can present so far provide\nsufficient jusCficaCon that Augmin does copurify with E. coli proteins as contaminants, and that\nthis can explain the presence of an addiConal density found in our cryo-EM images.\nMinor concerns\n1. The secGon ‘cryo-EM imaging of Augmin reveal dynamic architecture and higher order\noligomers’ is confusing. It might be helpful to add a cartoon to show the referred structural\ndomains.\nThis is a helpful suggesCon, and we added a cartoon diagram in the revised figure.\n2. Figure 1Ev is not menGoned in the text.\nWe thank the reviewer for catching the error. The reference is for Figure 1E. We fixed this in the\nrevised manuscript.\n3. It will be informaGve to show the raGo of each 2D-class average shown in Figure 1E.\nWe added the percenCle informaCon for each of the class averages in Figure 1E. –\n4. Line 263. I think ‘opposite topologies’ is a confusing term.\nWe have revised the sentence to improve clarity.\n5. The secGon “Augmin hetero-octameric structure stabilized by long and short-range coiled-\ncoil interacGons” is repeGGve. I suggest the authors revise these paragraphs.\nWe revised the paragraph to focus down the details and remove the repeCCon.\n6. Figure 5 is very confusing with all the colors.\nWe have revised Figure 5 to dim the colors of the Augmin subunits. We a dimmer tone colored\naugmin complex in the revised figure 5, which is necessary for the presentaCon otherwise it is\nhard to see which subunit interacts in XLMS data.\nReviewer #3 (Remarks to the Author):\nIn the paper enGtled “Cryo-EM structures of the Plant Augmin reveal its intertwined coiled-\ncoil assembly, anGparallel dimerizaGon, and NEDD1 binding mechanisms,” the authors have\ndetermined cryo-EM structures that lead to a complete de novo model of the coiled-coil\nassembly of the plant Augmin complex. They employed XL-MS approaches to validate these\nfindings. In their models, Augmin forms anGparallel dimers through conserved interfaces in its\nextended region. The authors also describe how the WD40 β-propeller domains of NEDD1\ndirectly bind to the Augmin V-juncGon, near the AUG6/7/8 microtubule (MT) binding site.\nAltogether, this paper presents a model in which NEDD1 promotes Augmin’s MT binding and\noligomerizaGon through downstream conformaGonal effects, ulGmately resulGng in the\nrecruitment of the γ-TuRC complex during MT branch formaGon.\nThe data presented are of high quality and of parGcular interest, especially given the cryo-EM\nanalysis of such elongated parGcles. The findings are well supported by clear figures and an\nappreciated supplementary video. Although the resoluGon achieved by cryo-EM was\nmoderate in some regions (10–20 Å), the integraGon of mulGple techniques (such as XL-MS\nand computaGonal modeling) enabled the authors to propose a robust model where NEDD1\nfacilitates Augmin’s MT interacGons.\nAs I am not an expert in cryo-EM, my primary focus was on the biochemical and cross-linking\nexperiments. However, the cross-linking data are difficult to evaluate since the raw files are\nnot available. AddiGonally, the exported search results do not align with the manuscript, and\nthus a thorough revision of this secGon is necessary prior to publicaGon.\nWe thank the reviewer for their helpful comments.\nWe had originally submieed our raw mass spec data to PRIDE, under the accession PXD060855.\nWe apologize for the inconvenience that the reviewer was not able to locate them; this must\nhave been due to a miscommunicaCon whereby we had provided the PRIDE reviewer login\ndetails upon submission, but the relevant informaCon may have not been accessible to the\nreviewer.\nIn any event, we have actually created a new PRIDE submission accession under the accession\nPXD065516. The access details are directly in the manuscript, but also reproduced here for your\nconvenience:\nToken: uleponAOr1sH\nUsername: reviewer_pxd065516@ebi.ac.uk\nPassword: cxd2zPo52d6l\nThe reason being is that in the Cme since our manuscript was originally submieed, a new set of\ndata reporCng standards have been described for XL-MS data (see\n10.1016/j.mcpro.2025.101024) which recommend providing mzIdentML files to facilitate data\nreuse and reanalysis. Our new PRIDE submission adheres to this standard.\nThe reviewer is correct also that search results (originally) did not align with the manuscript.\nWe discovered that there was an error from using Chimera to visualize crosslinks and XiView\nbecause the PDBs have missing residues (where there is missing density) but all crosslinking\ndata assign residue numbering based on the full-length sequences. This has since been recCfied\nin the revised Figure 5. Now, the crosslinks shown in the Figure 5 and Figure S11-S12, in the\nsupplemental excel file Data S1 (which provides all unique residue pairs), and in the search\nresults (which is on PRIDE) are all consistent.\nPoints to be addressed by the authors:\n• The authors state that they idenGfied 209 cross-links (XLs) within the Augmin hetero-\noctamer. However, amer extracGng all idenGfied XLs from the provided .csv file, and removing\nduplicates and decoys, I arrive at a total of 191 unique XLs. Could the authors clarify the origin\nof the reported number 209?\nWe have since reanalyzed all the crosslinking data, and fixed a serious error in which there was a\nmismatch between the residue numbering between the PDB and XiFDR/XiView. The new, final,\ncounts are 216 unique residue-pairs in the hetero-tetramer and 115 unique residue-pairs in the\nhetero-octamer. These 216 and 115 crosslinks are enumerated in Data S1. We have ensured\nthat decoys and duplicates are removed.\n• The two exported datasets differ significantly: one appears to be a raw export, including\ndecoy and reverse database results, while the other seems to be a pre-processed version.\nCould the authors please provide a detailed descripGon of the data processing pipeline and\nensure consistent exports for both the tetrameric and octameric forms?\nWe thank the reviewer for poinCng out the confusion. Our supplemental data file now provides\nfiltered unique residue-pairs, which is the form that would probably be the easiest for other\nresearchers to understand and work with.\nHere is the detailed descripCon of the data processing pipeline, reproduced from the text: RAW\nfiles were first converted and recalibrated using the xiSEARCH preprocessing pipeline python\nscript. The recalibrated MGF files were then searched with xiSEARCH 1.8.6 for crosslinked\npepCdes. Search parameters were used as following: MS1 mass tolerance, 6 ppm; MS2 mass\ntolerance, 10 ppm; allowed maximum number of missed cleavages, 4; minimum pepCde length,\n6. Crosslinks were searched based on the modificaCons at Lys and N-term (preferred),\nmodificaCons at Ser, Thr, and Tyr were also allowed with lower priority. CarbamidomethylaCons\n(+57.021464 Da) on Cys were enforced as fixed modificaCons, oxidaCons (+15.99491463 Da) on\nMet were allowed as variable modificaCons. Search results were filtered in xiFDR 2.3.2 at the\nresidue pair level to an FDR of 5%. The boost funcCon was enabled between residue pairs, and\nthe rest of the sewngs were default.\n• In Figure S11, the model contains significantly fewer XLs than the number of XLs reportedly\nidenGfied, despite the capGon staGng: “Augmin model presented in ribbon format with the\nobserved crosslinks generated in tube format.” The model clearly does not contain ~200 or\neven ~100 crosslinks. It appears that long-distance XLs (>30 Å) were excluded from the\nvisualizaGon, as none are visible. If this is the case, this filtering criterion must be clearly\nstated in the figure capGon.\nThe qualitaCve inconsistency that the reviewer is commenCng on was another unfortunate\nconsequence of the misnumbering error described before and now fixed. In the current version\nof the manuscript, we are much more explicit that we are only showing the crosslinks that fall\nwithin 35 Å. To ensure that this is clear, it is menConed in a piece of text embedded in the\nfigure itself (e.g., “216 crosslinks, 122 shown (<35 Å)”), in the figure legend, (“In total, 216\nunique residue pairs were idenCfied, of which the 122 with Cα-Cα distances less than 35 Å are\ndisplayed”), and in how it is discussed in the text.\n• The authors analyzed how many crosslinks from the hetero-octameric Augmin complex\n(AUG1–8) can be explained when allowing crosslinks to span across assemblies in the anG-\nparallel dimer model. While this is a valuable analysis, why is it limited to the octameric\nassembly, when the tetrameric form (Figure S11E) also shows four homo-inter XLs, compared\nto only two in the octameric form? This comparison should be expanded or explained.\nThe single parCcle cryo-EM imaging suggests that the AUG1,3,4,5 assembly does not form anC-\nparallel dimers, while the AUG1,2,3,4,5,6,7,8 assembly forms significant dimers. The structural\nexplanaCon for this stems from the AUG3,5-foldback zone being disordered as in AUG1,3,4,5\nbut not AUG1,2,3,4,5,6,7,8. Thus, we wanted to use the XLMS data to explain these the\norganizaCon of this anCparallel dimer.\n• Line 367: The statement that “many XLs are found, suggesGng the region is folded but\nflexible,” is not necessarily accurate. A high number of XLs may also result from an unfolded\nregion that is spaGally close due to the folding and stabilizaGon of other parts of the complex.\nThis interpretaGon should be reconsidered or rephrased.\nWe thank for the reviewer poinCng out the statement. This statement has been deleted, and\nindeed the whole XL-MS secCon has been rewrieen in the revised manuscript.\n• Line 382: The authors claim that “plots of our detailed XL-MS crosslinks on the de novo\nAugmin model are fully consistent with the criGcal and disGnguishing features of our\nstructural model.” However, this seems overstated, given that many XLs exceed the expected\nBS3 crosslinking distance (typically around 35 Å, with some reported exceeding 100 Å). This\nstatement should be tempered and clarified, possibly acknowledging model limitaGons or\nalternaGve explanaGons.\nYes, there are indeed many long-distance crosslinks, parCcularly for the hetero-tetramer, and\nwe agree with the reviewer that the original language menConed was hyperbolic. The secCon\nhas been rewrieen, and is more conservaCve in tone in the revised manuscript. We do believe\nthat the significant increase in the percentage of crosslinks that fall within the 35 Å cutoff for\nthe hetero-octamer provides evidence that the hetero-tetramer is more dynamic, which we\nhave several lines of evidence for. This is stated explicitly in the text:\n“That a higher percent of the crosslinks comports with the cryo-EM model suggest the octamer\nis a less dynamic assembly, consistent with our biochemical studies (Figure 1; Figure S1).”\n• Since “mass spectrometry also idenGfied several contaminaGng β-barrel–containing\nproteins” that copurify with Augmin through mulGple steps (as menGoned in the\nSupplementary list), these proteins should have been included in the database search used\nfor the XL-MS analysis. IdenGfying binding sites between these β-barrel proteins and Augmin\ncould have been helpful for structural interpretaGon. If insufficient XLs were obtained from\nco-purified complexes, the authors might consider performing a dedicated XL-MS experiment\non the complex formed between the full Augmin hetero-octamer (AUG1–8) and the NEDD1-\nWD40 β-propeller domain.\nWe have performed two versions of the XL-MS analysis. One in which only the Augmin subunits\nare included in the database search, and one in which bacterial contaminant proteins are\nincluded (46 for hetero-tetramer and 42 hetero-octamer respecCvely, based on contaminants\nwith the highest spectral matches in the linear search (Figure S12)). In the searches that include\nonly Augmin, we found 216 and 115 unique crosslinks respecCvely; in the searches with\nexpanded databases these go down to 163 and 89.\nOn the balance, we consider the results from the more restricted search database more\nauthoritaCve because they generated more idenCficaCons, and because the crosslinker we used\n(BS3) is non-cleavable and hence was not designed to be used for a larger database search.\nHence, the files we deposit to PRIDE, the supplemental data, and the figures reflect this version\nof the search.\nWe do think that it is useful to include the list of potenCal co-purifying proteins and the\ncrosslinking map including bacterial contaminants in the paper, and we have done so (in the\nform of Figure S12 and Data S1). We think these provide preliminary data to assign the\naddiConal density to RplB b-barrel ribosomal protein. But we do not think that they should be\nthe basis for the “primary” structural dataset of the XL-MS studies.\nMinor issues to be addressed:\nLine 129: Figure 1A does not illustrate the presence or absence of conserved α-helices; it only\nshows the construct length and the posiGon of the tags.\nWe have revised the text to correct this and removed the figure citaCon and added a reference\ncitaCon.\nSimilarly, Line 139: Figure 1A does not provide any relevant informaGon regarding the\nstatement “Mass spectrometry confirms each Augmin subunit is purified in the assembly and\nthat the AUG6 C-terminal region is prone to degradaGon with mulGple polypepGde bands.”\nThis should be clarified or corrected.\nWe revised the figure callout to be figure S1E for the statement above, which shows the\npercenCle coverage of each of the subunits based on mass spec results.\nLine 141: The manuscript references mass spectrometry data with “(Supplementary list file),”\nbut the actual data are missing. Please ensure that this file is included.\nWe have provided Data S1 which enumerates all idenCfied crosslinks\nLine 348: It is unclear which complex (tetramer or octamer) the reported values “50% (105 out\nof 209)” and “70% (80 out of 115)” refer to. This should be explicitly stated.\nWe have revised the text to clarify which complex is which and address this"}, {"round": 2, "reviewer_comments": {"reviewer_1": "Regarding my minor point #1, I continue to believe that the authors should revise the text in line 82. Additionally, identifying RplB as the globular density in the cryo-EM map is not very convincing. If RplB tightly binds to augmin, it should be possible to detect the RplB band on SDS-PAGE and easily identify it by traditional mass spectrometry along with augmin subunits (line140-141).", "reviewer_2": "The authors have addressed my questions. I have no further comments.", "reviewer_3": "I would like to thank the authors for their efforts in addressing my previous remarks and for improving their data analysis, particularly concerning the XL-MS part of their paper entitled “Cryo-EM structures of the Plant Augmin reveal its intertwined coiled-coil assembly, anGparallel dimerization, and NEDD1 binding mechanisms.” I also appreciate their willingness to make the raw data easily accessible, thereby ensuring transparency and open access to their research.\nAfter this revision, the XL-MS section is now much clearer, easier to follow, and aligns better with the structural models proposed for the Augmin assemblies.\nNevertheless, before this work can be published—as it indeed deserves—there remain some discrepancies between the raw data and the data presented in the paper that must be resolved. Specifically, when examining the raw exports from the XiSearch search engine after FDR validation (i.e., links_xiFDR2.3.2.csv files), I could not find several cross-links (XLs) reported in the paper’s dataset (Dataset S1 Source file XLS). Neither the total number of XLs nor the specific XL amino acid pairs match.\nInitially, I considered that redundancy might explain the differences; however, after confirming that there were no duplicates or decoys, discrepancies persisted. For example, in the tetrameric XL dataset, the paper reports 216 cross-links (consistent with the text), whereas the T8_15_combined_Links_xiFDR2.3.2.csv file in the PRIDE repository contains 277. Similarly, in the octameric XL experiment, 115 cross-links are reported in the paper but 172 appear in the raw export from PRIDE. If the authors applied additional filters beyond the FDR, these criteria should be explicitly stated and justified.\nMoreover, despite some cross-links being absent from the raw export, others appear in the dataset presented in the paper but are not found in the original data. When comparing the octameric XL lists, I identified 26 cross-links that are present in the paper but absent from the raw export. While it is understandable that certain XLs might be excluded due to stricter filtering (which should also be explained), it is unclear how non-identified XLs could appear in the published dataset. The authors should correct this inconsistency.\n________________________________________\nMinor Comments\nLines 391–394:\n“Because the dimer model lacks the V-junction, only 59 of the 115 cross-links could be mapped to the dimer model, but of those 59, 32 had even shorter Cα–Cα distances in the anti-parallel dimer model, suggesting the presence of Augmin assemblies in the anti-parallel dimer state in solution (Figure 5C; Figure S11C,F).”\nThis sentence is overly result-oriented. The fact that a cross-link exhibits a shorter Cα–Cα distance in one model compared to another does not necessarily imply stronger support for that model, especially when both fall within the range of a cross-linker. The probability of forming a cross-link (crosslinkability) is determined by multiple structural and dynamic factors. For a discussion of such criteria, please refer to:\nFilella-Merce, I. et al. (2020). “Quantitative Structural Interpretation of Protein Crosslinks.”"}, "author_response": "Dear editor and reviewers,\nWe are submitting a further revised version of our manuscript #NCOMMS-25-15494\nentitled “Cryo-EM structures of the Plant Augmin reveal its intertwined coiled-coil\nassembly, antiparallel dimerization and NEDD1 binding mechanisms” We thank\nreviewers 1 and 3 for their attention to detail on the mass spectrometry data. We have\nmade a few revisions to the text and to the supporting data file to rectify the matters that\nthey have raised.\nPlease see a point-by-point response below.\nOur responses are in blue, and the small changes to the text are bold-faced.\nReviewer #1 (Remarks to the Author):\nRegarding my minor point #1, I continue to believe that the authors should revise the\ntext in line 82. Additionally, identifying RplB as the globular density in the cryo-EM map\nis not very convincing. If RplB tightly binds to augmin, it should be possible to detect the\nRplB band on SDS-PAGE and easily identify it by traditional mass spectrometry along\nwith augmin subunits (line140-141).\nWe agree that the assignment of the mystery data to RplB is at best, our best guess. It\nis supported by XL-MS and “traditional” mass spectrometry, as the reviewer suggests it\nought to have been.\nWe have made a few modifications to the text to make it clearer that the assignment is\ntentative, and that RplB-derived tryptic peptides were indeed robustly identified as an\nabundant contaminant in the “traditional” linear peptide mass spec searches.\n“The V-junction consistently possesses a high electron density spot. Mass spectrometry\nanalysis is suggestive that this interactor is RplB…”\n“A globular density, represen4ng a bright spot residing inside the V-junc4on, poten&ally\ncorresponds to the ribosomal protein, RplB. Mass spectrometry on the Augmin pulldowns\nshows RplB to be an abundant contaminant with 518 pep&de-spectrum matches, and\nmoreover RplB crosslinks to AUG5 (see below; Figure S12). A b-barrel domain was placed in this\ndensity but was excluded from our model building and interpreta4on (Figure 1E, panel V; Figure\nS5B, G).”\nReviewer #2 (Remarks to the Author):\nThe authors have addressed my questions. I have no further comments.\nReviewer #3 (Remarks to the Author):\nI would like to thank the authors for their efforts in addressing my previous remarks and\nfor improving their data analysis, particularly concerning the XL-MS part of their paper\nentitled “Cryo-EM structures of the Plant Augmin reveal its intertwined coiled-coil\nassembly, anGparallel dimerization, and NEDD1 binding mechanisms.” I also\nappreciate their willingness to make the raw data easily accessible, thereby ensuring\ntransparency and open access to their research.\nAfter this revision, the XL-MS section is now much clearer, easier to follow, and aligns\nbetter with the structural models proposed for the Augmin assemblies.\nNevertheless, before this work can be published—as it indeed deserves—there remain\nsome discrepancies between the raw data and the data presented in the paper that\nmust be resolved. Specifically, when examining the raw exports from the XiSearch\nsearch engine after FDR validation (i.e., links_xiFDR2.3.2.csv files), I could not find\nseveral cross-links (XLs) reported in the paper’s dataset (Dataset S1 Source file XLS).\nNeither the total number of XLs nor the specific XL amino acid pairs match.\nInitially, I considered that redundancy might explain the differences; however, after\nconfirming that there were no duplicates or decoys, discrepancies persisted. For\nexample, in the tetrameric XL dataset, the paper reports 216 cross-links (consistent with\nthe text), whereas the T8_15_combined_Links_xiFDR2.3.2.csv file in the PRIDE\nrepository contains 277. Similarly, in the octameric XL experiment, 115 cross-links are\nreported in the paper but 172 appear in the raw export from PRIDE. If the authors\napplied additional filters beyond the FDR, these criteria should be explicitly stated and\njustified.\nWe thank the reviewer for paying close attention to this issue. The reason for the\ndiscrepancy was because Dataset S1 only reported on the crosslinks that could be\nmapped to residues that were included in the cryoEM reconstructed model. On the\nother hand, the outputs form xiFDR include all crosslinks that were confidently\nidentified, regardless of whether they could be mapped to the structure or not.\nTo rectify the issue, Dataset S1 has been modified. Tabs 1 and 2 (which provide a list\nof all the unique crosslinks in AUG1,3,4,5 and AUG1,2,3,4,5,6,7,8 respectively) now\nprovides a comprehensive list of crosslinks (277 and172 respectively). It is obvious\nfrom the spreadsheet that the additional 61 and 57 crosslinks (at the bottom of the lists)\nare not in the model because they lack values in columns K, L, and M which assigns the\ncrosslink a distance and PDB chain identifiers.\nTo make sure that this exclusion of some of crosslinks is clear to the reader, we have\nslightly amended the language in the text accordingly:\n“Plotting the Ca-Ca distances for the 216 crosslinks that can be fit within the Augmin\nhetero-tetramer (AUG1,3,4,5) cryo-EM model reveals that 122 (56%) of these crosslinks\nfall below a 35 Å cut-off, and are shown in Figure 5A (see also Figure S11A, D, G).”\n“Plotting the Ca-Ca distances for the 115 identified crosslinks that can be fit within the\nAugmin hetero-octamer (AUG1,2,3,4,5,6,7,8) cryo-EM model reveals that 85 (74%) of\nthese crosslinks fall below a 35 Å cut-off, and are shown on the model in Figure 5B\n(see also Figure S11B, E, H).”\nMoreover, despite some cross-links being absent from the raw export, others appear in\nthe dataset presented in the paper but are not found in the original data. When\ncomparing the octameric XL lists, I identified 26 cross-links that are present in the paper\nbut absent from the raw export. While it is understandable that certain XLs might be\nexcluded due to stricter filtering (which should also be explained), it is unclear how non-\nidentified XLs could appear in the published dataset. The authors should correct this\ninconsistency.\nAfter carefully reviewing the files, we see that the reviewer made the small mistake of\nnot considering the possibility that the order of which crosslink site is called site1 and\nwhich crosslink site is called site2 can sometimes get permuted between xiFDR and\nxiView (the latter was used to generate the output file that became adapted into\nDataset1).\nTo show that we have done our due diligence and can account for every single\ncrosslink, we have supplied two supplemental spreadsheets for review purposes only\n(not to be published). They are called tetrameric_xlms and octameric_xlms.\nIn tetrameric_xlms, we have two tabs. The first tab, in columns A–M, is a reproduction\nof Dataset S1’s report for AUG1,3,4,5. In column N, we provide an identifier for each\ncrosslink by concatenating protein1, residue1, protein2, residue 2, and in column O, we\nprovide the alternative identifier for that crosslink if site1 and site2 were interchanged.\nThes second tab provides the xiFDR output for AUG1,3,4,5 in columns A-AP, and in\ncolumns AQ and AR we provide identifiers for each crosslink using the same format,\nagain allowing for interchange. In column P of the first tab, we use an Excel formula\nthat asks whether this crosslink in Dataset S1 is also in the xiFDR report (in either the\nsame or altered order). All 277 are found in the xiFDR report. In column AS of the\nsecond tab, we use the same Excel formula to ask if this crosslink in the xiFDR report is\nalso in Dataset S1. All 277 are found in Dataset S1.\nThe file called octameric_xlms does the same quality control check for\nAUG1,2,3,4,5,6,7,8 and also shows 100% consistency.\n________________________________________\nMinor Comments\nLines 391–394:\n“Because the dimer model lacks the V-junction, only 59 of the 115 cross-links could be\nmapped to the dimer model, but of those 59, 32 had even shorter Cα–Cα distances in\nthe anti-parallel dimer model, suggesting the presence of Augmin assemblies in the\nanti-parallel dimer state in solution (Figure 5C; Figure S11C,F).”\nThis sentence is overly result-oriented. The fact that a cross-link exhibits a shorter Cα–\nCα distance in one model compared to another does not necessarily imply stronger\nsupport for that model, especially when both fall within the range of a cross-linker. The\nprobability of forming a cross-link (crosslinkability) is determined by multiple structural\nand dynamic factors. For a discussion of such criteria, please refer to:\nFilella-Merce, I. et al. (2020). “Quantitative Structural Interpretation of Protein\nCrosslinks.”\nWhile we agree that many factors influence the propensity for two residues to form a\ncrosslink (with distance only being one), we do maintain that this comparison is a\nreasonable one because it compares distances for the same residue pair. Hence, other\nfactors that contribute to crosslinkability (like residue chemistry, solvent exposure) for a\ngiven residue pair is nearly identical between the two structural models\n(monomer/dimer) we’re comparing.\nThat said, we agree with the reviewer in spirit, and so have softened the language used\nin the text. Now we say that including the anti-parallel dimer state allows to better\ncapture some of the crosslinking data instead of asserting that it directly implies that the\nanti-parallel dimer is physically present:\n“Because the dimer model lacks the V-junction, only 59 of the 115 crosslinks could be\nmapped to the dimer model, but of those 59, 32 had even shorter Ca-Ca distances in\nthe anti-parallel dimer model, suggesting that inclusion of the anti-parallel dimer\nstate allows us to better capture some of the crosslinking data.”"}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors have addressed my questions. I have no further comments.", "reviewer_3": "I would like to thank the authors for the constructive exchanges regarding their manuscript.\nThe authors have satisfactorily addressed my questions, and I have no further comments."}, "author_response": ""}]

Decision letter