K2 direct detector

Micrographs were collected on a low-base Titan electron microscope (Thermo Fisher) operated at 300kV and equipped with a K2 direct detector (Gatan) using the Leginon automated software [61 (link)]. The micrographs were collected at a nominal magnification of 27,500x, resulting in a final pixel size of 1.32 Å per pixel at the sample. Twenty frames of 300ms each were collected at a dose rate of 8 e⁻ per pixel per s, with a total dose of 28 e⁻/Ų.
Micrographs were processed as previously described [12 (link),34 (link),62 (link)]. Briefly, frame alignments were performed using MOTIONCORR [63 (link)], and CTF estimation using CTFFIND4 [64 (link)]. MTs were manually identified and extracted using overlapping square boxes with an edge length of ∼675 Å that were spaced ∼80 Å apart, corresponding to approximately on turn of tubulin dimers. Each of these boxes was treated as an independent, single particle [65 (link)] during sorting by protofilament number and initial 3D alignment using EMAN2 [66 (link)] with models of 12, 13, 14, and 15 protofilament MTs [67 (link)]. The total number of dimers imaged for each MT protofilament number was calculated by multiplying the number of boxes that were assigned to the model by its protofilament number.

Data were collected on a Titan Krios G3 (Thermo Fisher) equipped with a K2 direct detector (Gatan) at 300 keV using the semi-automated data acquisition software EPU (Thermo Fisher). 40 frames with a dose of 1.09 ^e−/Ų per frame were collected in a defocus range of −0.4 to −3.5 µm. Magnification settings resulted in a pixel size of 1.045 Å/pixel. Frame alignment was executed with MotionCor2 (56) and the estimation of the contrast transfer function (CTF) was performed with Gctf (57).