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. 2020 Nov 30;9:e61481. doi: 10.7554/eLife.61481

Figure 2. Kinesin-binding protein (KBP) conformationally adapts to bind KIF15’s motor domain via both subdomains.

(a) Model of the KBP–KIF15_MD6S complex (ribbon representation) displayed in experimental cryo-electron microscopy density. The N-terminal (olive) and C-terminal (gold) subdomains and the linker helix (black) are shown in KBP, while kinesin is coloured in magenta. Semi-transparent density is coloured regionally as per the fitted model and additional density for the linker loop is shown in semi-transparent black. (b) The same as panel a, but rotated 180° around the axis indicated. (c) The KBP-alone model (light grey ribbons) was superimposed on the KBP–KIF15_MD6S model (opaque ribbons) using Chimera’s matchmaker (Pettersen et al., 2004). Colouring and view as in panel b. (d) RMSD in Å for KBP comparing KBP–KIF15_MD6S and superimposed KBP-alone models as in panel c, shown on KBP from the KBP–KIF15_MD6S model. Parts of the KBP model coloured black are disordered/missing in the KBP alone model. The KIF15_MD6S is shown in transparent magenta.

Figure 2.

Figure 2—figure supplement 1. Kinesin-binding protein (KBP)–KIF15_MD6S reconstruction resolution estimation and 2D class analysis of KBP–KIF1A_MD and KBP–KIF15_MD complexes.

Figure 2—figure supplement 1.

(a) KIF15_MD6S MT-activated steady-state ATPase velocity plotted as a function of [MT]. Data were fit to a Michaelis–Menten kinetic (pink curve) yielding values for kcat = 2.9 ± 0.5 s−1 and K0.5,MT = 4.8 ± 1.4 μM; R2 = 0.97, which are very similar to previously published values for KIF15_MD of kcat = 2.1 s−1 and K0.5,MT = 3.1 μM (Klejnot et al., 2014). (b) Gold-standard Fourier shell correlation (FSC) curves between independent masked, unmasked, phase-randomised, and corrected half-maps (Chen et al., 2013) of the KBP–KIF15_MD6S complex as calculated by RELION v3.0 (Zivanov et al., 2018). The resolution at the ‘gold-standard’ 0.143 FSC cutoff is 6.9 Å. (c) Local resolution as calculated by RELION v3.0, shown on the same view as in Figure 2a with coloured density corresponding to the local resolutions indicated in the key. (d) Selected RELION v3.0 (Zivanov et al., 2018) 2D classes of KBP–KIF15_MD6S (left) and KIF1A_MD–KBP (four to the right). Densities for the kinesin motor domain and KBP are pseudo-coloured pale magenta and pale orange respectively. Classes have been in-plane rotated such that KBP is seen from roughly the same orientation. Note poor resolution and a variable relative position in the KIF1A_MD. (d) A representative subset of KBP–KIF15_MD6S complex 2D classes, showing multiple orientations.