Three-dimensional reconstruction of Mal3-143-decorated A-lattice microtubule. (a) Side view of the microtubule. (plus end upward). (b) End-on projection seen from the plus end. Mal3 density is highlighted in yellow. Asterisks show where Mal3 (m) almost touches a tubulin subunit in the neighboring protofilament. Subunits t1 and t2 may represent α- and β-tubulin, respectively.

Model of Mal3 promotion of A-lattice assembly. In vitro tubulin heterodimers will form oligomers that may either continue to assemble or may dissociate again. We propose that Mal3 binds to and stabilizes A-oligomers better than B-oligomers, thus promoting their growth into larger complexes and seeding a future microtubule with a high proportion of A-lattice contacts. As a microtubule grows, Mal3 binds tightly to the tip, which might correspond to an extension with a similar structure to the oligomer seeds. Mal3 binds only weakly to the closed microtubule lattice. White subunits, α-tubulin; green subunits, β-tubulin. Stably bound Mal3 monomers are shown in magenta. More weakly bound Mal3 is shown in blue. γ-tubulin complexes (orange and yellow)^27,28 may initiate pure A-lattice assembly in vivo by interacting with both α- and β-tubulin²⁹.

Mal3 promotion of microtubule assembly in vitro. (a) Left, domain structure of Mal3. CH and EB1-like C-terminal motif domains are indicated by green and orange, respectively. Boundaries for each domain are indicated by residue numbers. The amino acid sequence for residues 130-150 is shown below and the three arginine residues are highlighted in red. Right, domain structure for the constructs used in this study. 6×His-tags are highlighted in blue. (b) Microtubule-polymerization assay by 90° light scattering at λ = 350 nm. All assays contained 4 μM S. pombe tubulin (heterodimer concentration). Colored lines indicate the added Mal3 monomer concentration; orange, 0 μM; green, 2 μM; brown, 4 μM; blue, 8 μM.

Microtubules assembled with Mal3 show mixed lattices. (a,b) Diagrams of microtubules with A- and B-lattices of tubulin heterodimer subunits, represented both as three-dimensional microtubules and as opened-out sheets. White subunits, α-tubulin; green subunits, β-tubulin; blue lines, the direction of the 3-start family of helices, common to both lattices, that arises because subunits in adjacent longitudinal protofilaments are staggered by 0.9 nm; yellow lines, the directions of the decoration patterns, different on both lattices, as shown in e and f. (c) Diffraction pattern from a cryo-EM image of a pure tubulin microtubule decorated with kinesin head domains. Red and orange lines show the B-lattice contributions to the diffraction pattern. Red lines, contribution of the near side of the microtubule; orange lines, contribution of the far side. (d) Cryo-EM image of a microtubule copolymerized with Mal3 and its corresponding (mixed AB) diffraction pattern. Blue and green lines show the A-lattice contributions to the diffraction pattern. Blue lines, near-side contribution; green lines, far-side contribution. (e,f) Negative-stain EM images of microtubules decorated with kinesin, showing a pure tubulin microtubule (e) and a microtubule copolymerized with Mal3 (f). The yellow lines highlight the rows of kinesin on the microtubule walls (see a,b.) (g) Diffraction pattern of image e. (h) Diffraction pattern of image f. (i) cryo-EM image of a microtubule copolymerized with Mal3 and its corresponding A-lattice diffraction pattern.

Mal3 binding to microtubules in vitro. (a) Binding of Mal3-143 to GMPCPP-stabilized S. pombe microtubules, as quantified by SDS-PAGE. The graph shows a single experiment and gave a fitted K_d of 2.4 ± 0.9 (±S.E.) μM. (b) Typical gel run for a pelleting assay. In this case, Mal3-143 (added concentrations, 0-12 μM, are indicated above the lanes) and 8 μM of S. pombe tubulin (Tb) heterodimers were copolymerized in the presence of 1 mM GTP or GMPCPP. s, supernatant fraction; p, pellet fraction. (Curves plotted for the samples with GTP are shown in Supplementary Fig. 2a.). (c) Binding of Mal3-308 to GMPCPP-stabilized S. pombe microtubules. The graph contains all the data points from three independent experiments. For the fitted curve, B_max 1.3 ± 0.14 (±S.E.) μM and K_d 0.93 ± 0.28 (± S.E.) μM (giving 95% confidence intervals of 0.98-1.6 μM for B_max and 0.35-1.5 μM for K_d). (d) Binding of 2.5 μM Mal3-308 to pig brain microtubules (MT), either after coassembly with tubulin (red) or after being mixed with preassembled GMPCPP-stabilized microtubules (blue). The data points shown are the result of one experiment. Two other experiments, using either GMPCPP or Taxol to stabilize the preassembled microtubules, gave similar differences from coassembled samples.