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. 2020 Aug 10;9:e58157. doi: 10.7554/eLife.58157

Figure 1. VASH1-SVBP efficiently detyrosinates and binds microtubules.

(A,B) Tubulin detyrosination assays of VASH1-SVBP in human cells. HeLa Tet-On cells were co-transfected with VASH1 and SVBP plasmids, and treated with 5 µM nocodazole (A) or 100 nM Taxol (B) for indicated times at 24 hr post-transfection. The cell lysates were blotted with the indicated antibodies. deY-tubulin, detyrosinated α-tubulin. Experiments were repeated three times with similar results. (C) Quantification of the relative detyrosination levels of α-tubulin in (A) and (B) (mean ± s.d., n = 3 independent experiments). Significance calculated using two-tailed student’s t-test; between control cells and cells treated with nocodazole or Taxol for the indicated time; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. (D) Microtubule pelleting assays showing the binding of VASH1-SVBP to recombinant human microtubules. S, supernatant; P, pellet. (E) Cryo-EM map of 14-protofilament, GMPCPP-stabilized microtubules decorated by the VASH152-310-SVBP complex. The catalytically inactive C169S mutant of VASH1 was used in the complex. The map is lowpass filtered to 4 Å. The microtubule seam is indicated by a red dashed line. α-tubulin, β-tubulin, VASH1, and SVBP are colored in green, cyan, blue, and orange, respectively. The same color scheme is used for all figures. The inset shows a close-up view of the boxed region. (F) Close-up view of the cryo-EM map in (E), viewed from the lumen. The α- and β-tubulin molecules can be distinguished by the length of the S9-S10 loop (boxed with red dashed lines), with the loop in α-tubulin being longer.

Figure 1.

Figure 1—figure supplement 1. Structure determination of VASH1-SVBP-decorated GMPCPP-microtubules.

Figure 1—figure supplement 1.

(A) A representative micrograph of VASH1-SVBP-decorated GMPCPP-microtubules. Scale bar, 50 nm. (B) Processing workflow for cryo-EM structure determination of VASH1-SVBP-decorated GMPCPP-microtubules. The 2D classes of poorly decorated microtubules (red outlines) were discarded whereas the classes belonging to efficiently decorated microtubules (green outlines) were selected for subsequent 3D classification. (C) Fourier shell correlation (FSC) curves of GMPCPP-microtubules decorated with VASH1-SVBP. The FSC curves of microtubules (top) and VASH1-SVBP (bottom) were calculated separately. The final resolution for the reconstruction was estimated by calculating the Fourier shell correlation (FSC) of a single tubulin heterodimer in a ‘good’ protofilament after pseudo-helical averaging, using the FSC = 0.143 criterion.