Microtubule Orientation Alignment Protocol

Having determined the relative polarity, the necessary step for combining is finding the relative φ rotation (rotation around the symmetry axis) between individual microtubules. This could be done by aligning each individual particle to a common reference, but would result in a large number of errors due to the low signal to noise ratio. We developed a method analogous to that of Zabeo et al.,^{25 (link)} where the microtubule average volumes are aligned together in order to find their relative φ rotation (Fig. S9 step 4). These φ rotations were then applied to their respective particles and aligned to a common reference. SVA was subsequently performed with volumes binned 4 and 2 times. Only sporozoite data were aligned with unbinned volumes; the final step was to replace volumes reconstructed with the whole sets of tilts by volumes reconstructed with tilts between ±24°. No rotation search was performed with the restricted tilt range. The resulting C1 EM map was anisotropic around the pseudosymmetry axis due to an uneven distribution of microtubule rotational orientations. To address this, particles were separated into classes by orientation and particles with the lowest cross correlation coefficients in the most abundant classes were removed. This reduced the number of particles from 24028 to 13263 in sporozoite and 8377 to 1851 in ookinete datasets.