Supplementary Figure 8: Reconstruction of the CAMSAP1 N1492A CKK mutant and analysis of the effect of CKK on MT polymerization.
From: A structural model for microtubule minus-end recognition and protection by CAMSAP proteins
(a) The asymmetric reconstruction of the CAMSAP1-N1492A CKK decorated 13pf paclitaxel-stabilized MT low-pass filtered to 15Ã… resolution shows extra densities (green) every 8nm corresponding to the CAMSAP1-N1492A CKK domain, which are absent at the seam similar to wild type (arrow).
(b) FSC curves utilizing the gold-standard noise substitution method (Chen, S. et al., Ultramicroscopy 135, 24-35, 2013) give an overall resolution estimate for the CAMSAP1-N1492A CKK-MT reconstruction of 4.8Ã….
(c) The averaged reconstruction of the CAMSAP1- N1492A CKK domain viewed from the MT surface contacting two β-tubulins and two α-tubulins at the intra-dimer, inter-protofilament interface. The CAMSAP1-N1492A CKK is colored as previously. α-tubulin is shown in light grey and β-tubulin is shown in dark grey. Cryo-EM density is shown as transparent light blue solid. Above, schematic.
(d) View from the plus end of the CAMSAP1-N1492A CKK decorated 13pf MT, showing details of the tubulin contacts of the N-terminus (red), helix α1, loop1 (magenta) and loop 7 (yellow), colored as in Supplementary Fig. 5e. The position of N1492A (brown) is also indicated (arrowhead). The experimental density is shown as blue transparent solid, with tubulin shown as grey ribbons fitted into the experimental density.
(e) Protofilament skew for representative MTs from each dataset is depicted as in Supplementary Figure 4a. The reduced skew produced by CAMSAP1-N1492A CKK-decorated MTs are compared to wild type CKK MTs and control kinesin decorated paclitaxel-stabilized MTs (datasets from Atherton, J. et al., eLife 3, e03680,2014). For CAMSAP1 CKK, n=16, for kinesin1 decorated MTs, n=16; for CAMSAP1-N1492A CKK-decorated MTs, n=15. Data represent mean ± SD. 1-way ANOVA, CAMSAP1 CKK N1492A vs CAMSAP1 CKK wild type, p<0.001;CAMSAP1 CKK N1492A vs kinesin-1 13pf p<0.05.
(f) Copolymerisation of tubulin with CAMSAP1 CKK domain yields few MTs but produces clumps of heterogeneous tubulin oligomers (left), while copolymerisation with CAMSAP-N1492A yields mainly MTs (right). Representative cryo-EM images of the products of each polymerisation reaction are shown.
See also Supplementary Table 2.
