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

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.

Fig. 1. Biochemical and structural characterization of A. thaliana Augmin assemblies.

A Schematic representation of plant Augmin hetero-octamer (AUG1,2,3,4,5,6,7,8) subunit organization, showing domain boundaries and purification tags. Note: AUG8 construct (residues 383–644) excludes the N-terminal MT binding domain (See Supplementary Fig. 1). B Biochemical validation of hetero-octameric Augmin complex. Left: SEC-MALS analysis confirming monodisperse assembly with predicted octameric mass (single run). Right: SDS-PAGE of purified complex showing all eight subunits (See Supplementary Fig. 1). AUG6^* marks a degradation product of AUG6. C Schematic of minimal Augmin hetero-tetramer (AUG1,3,4,5) subunit organization, including domain boundaries and purification tags. D Biochemical validation of hetero-tetrameric complex. Left: SEC-MALS analysis demonstrating monodisperse assembly with tetrameric mass (Single run). Right: SDS-PAGE confirming presence of four subunits. E Cryo-EM 2D-class averages revealing Augmin complex architecture. Top panel: Monomeric AUG1,2,3,4,5,6,7,8: 40 nm tuning fork structure. Second panel: Monomeric AUG1,2,3,4,5,6,7,8: V-junction focus with extended region. Third panel: Dimeric AUG1,2,3,4,5,6,7,8: Two extended regions, single V-junction. Fourth panel: Dimeric AUG1,2,3,4,5,6,7,8: Two extended regions, two V-junctions. Fifth panel: Focused V-junction from AUG1,2,3,4,5,6,7,8: 24-nm V-junction and stem regions. Bottom Panel: Monomeric AUG1,3,4,5: 23 nm extended region.

Fig. 2. Single particle Cryo-EM structures and models of Augmin assemblies.

A Cryo-EM reconstructions of the AUG1,2,3,4,5,6,7,8 V-junction and stem. Left: 7.3-Å structure with segmented model of AUG1,2,3,4,5,6,7,8 (closed-state AUG6,7 CH-domain dimer). Right: 10 Å structure with open-state AUG6,7 CH-domain dimer (See Supplementary Figs. 2–4). B Model analysis. Left: AUG1,2,3,4,5,6,7,8 subunit organization in V-junction and stem. Right: Conformational comparison of closed (red) and open (blue) states. Inset: Vector representation of conformational transition. C 3.7 Å reconstruction of AUG1,3,4,5, extended region with segmented model (See Supplementary Figs. 2–5). D Detailed subunit organization of AUG1,3,4,5 extended region model. E Composite model of complete AUG1,2,3,4,5,6,7,8 complex derived from combined maps and models (See Supplementary Fig. 5A–C).

Fig. 3. Architectural coiled-coil organization and conservation of the Augmin assembly.

A Ribbon diagram highlighting individual subunit folds and organization. B Subcomplex interaction analysis. Left: AUG3,5 heterodimer with interaction footprints. Right: AUG1,4 and AUG2,6,7,8 subcomplexes with corresponding interfaces. Insets: Topological organization of each subunit. C Complete Augmin hetero-octamer assembly showing structural domains. D Electrostatic surface potential map highlighting functional regions (Supplementary Fig. 9A–C). E Surface conservation analysis across plant, animal, and insect (Supplementary Fig. 9F–H).

Fig. 4. Cryo-EM structure and analysis of Augmin antiparallel dimer assembly.

A Top: 2D class averages showing ~65 nm antiparallel organization of extended domains extracted in 450 pixels box at 1.76 Å/pixel. Bottom: Low-resolution reconstruction with focused refinement of dimerization interface. B Detailed structural analysis of extended region dimer. Left: Side view of segmented reconstruction with colored subunit organization. Middle: 90° rotated view showing subunit arrangement. Right: Isolated model highlighting subunit organization at dimer interface. C Dimer interface analysis showing protomer organization. Red/blue: individual protomers. Yellow: Interface region between protomers. D Conformational changes in dimer formation. Splayed view of protomers showing AUG1,3,4,5 organization. Interface zones highlighted in red at foot and belly regions. Arrows indicate domain movements during dimerization. E Monomer-to-dimer transition analysis. Left: Single protomer structure. Middle: Overlay of monomeric and dimeric states. Right: Vector representation of conformational changes in tripod, belly, and leg regions.

Fig. 5. XL-MS validates features of Augmin hetero-tetramer, Augmin hetero-octamer, and antiparallel Augmin dimer.

A Crosslinks identified in AUG1,3,4,5, illustrated in the context of the cryo-EM structural model of AUG1,3,4,5. In total, 216 unique residue pairs were identified, of which the 122 with Cα-Cα distances less than 35 Å are displayed. B Crosslinks identified in AUG1,2,3,4,5,6,7,8 illustrated in the context of the cryo-EM structural model of AUG1,2,3,4,5,6,7,8. In total, 115 unique residue pairs were identified, of which the 85 with Cα-Cα distances less than 35 Å are displayed. C Crosslinks identified in the AUG1,2,3,4,5,6,7,8 illustrated in the context of the cryo-EM structural model of the anti-parallel Augmin dimer. The 32 crosslinks which have shorter Cα-Cα distances in the context of the anti-parallel dimer compared to the monomer are shown.

Fig. 6. Biochemical reconstitution and structure NEDD1 WD β-propeller-Augmin complex.

A NEDD1 domain organization. N-terminal WD40 β-propeller domain (termed NEDD1-WD β-propeller). C-terminal helical domain (Note: Only WD β-propeller domain successfully purified). B Complex formation analysis: AUG1,2,3,4,5,6,7,8 binds NEDD1-WD β-propeller and while AUG1,3,4,5 shows no binding (See Fig. S12C–H). C SEC analysis of complex formation. Red: AUG1,2,3,4,5,6,7,8 alone. Blue: AUG1,2,3,4,5,6,7,8 with NEDD1-WD β-propeller. Green: NEDD1-WD alone. D Biochemical validation of SEC analysis in (C) by SDS-PAGE showing co-migration of NEDD1-WD with AUG1,2,3,4,5,6,7,8 and comparison with AUG1,2,3,4,5,6,7,8 alone (N = 2). E Cryo-EM 2D-class average comparison NEDD1-WD β-propeller binding to Augmin. Top: AUG1,2,3,4,5,6,7,8 V-junction-stem with β-barrel density. Bottom: AUG1,2,3,4,5,6,7,8-NEDD1-WD β-propeller complex. F Structural characterization of complex. Left: 12 Å Cryo-EM segmented reconstruction with fitted models. Right: Ribbon representation of complex. G 7.3-Å model showing β-barrel density bound to V-junction stem. Note the marked conformational change. H Comparative analysis of V-junction. Top: Segmented maps of complex vs. AUG1,2,3,4,5,6,7,8 alone. Bottom: Atomic models of NEDD1-WD and β-barrel regions. I NEDD1-WD-β propeller interface analysis. Right: Surface views of Augmin interaction site. Left: Conservation analysis across species.

Fig. 7. Coevolutionary analysis and direct coupling analyses support the Augmin hetero-octameric assembly structure and its interface with NEDD1-WD β propeller.

A Circular summary of eukaryotes with Augmin subunit(s) and NEDD1. Concentric circles encode taxonomy (center—kingdom; middle—class. The outer rim annotates conservation of the eight Augmin subunits and NEDD1 across species (pink—conservation; light cyan—absence of one or more subunits). A complete evolutionary tree is shown in Supplementary Fig. 15A. B Line-based co-evolutionary representation of Augmin subunits and NEDD1 across eukaryotes. Each line represents a unique species. Line color code: yellow—protein is absent; green—protein is present; purple—complete set (all eight Augmin subunits plus NEDD1 in that species). C Structural model of the Augmin hetero-octamer displaying DCA pairs as bars across Augmin subunits. Pairs are colored by significance (red: p ≤ 0.05, blue: 0.05 <p ≤ 0.1; black: 0.1 <p ≤ 0.2). The p-values 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. Full pair list is in Supplementary Table 3. D Circular view of the sequences in Augmin subunits with the DCA pairs colored as in (C). E Augmin hetero-octamer V-junction model displaying the NEDD1-WD β-propeller and its DCA pairing (black bars) to AUG2,5,6,8, as detailed in Supplementary Table 5.

Fig. 8. Mechanistic model of Augmin-NEDD1-MT interactions and MT branch nucleation.

A MT lattice binding modes. Left: NEDD1-WD complex with closed AUG6,7 CH-domains. Center-left: β-barrel density impact on MT binding. Center-right: Open CH-domain configuration. Right: NDC80/Nuf2 CH-domain reference structure views shown in a side and a 90° rotated orientation. B Proposed mechanism for Augmin function. Left: Transition from diffuse to NEDD1-WD-bound state. Middle: Formation of anchored complex and dimerization. Right: γ-TuRC recruitment and MT branch nucleation.