Low-dose movies were collected automatically using EPU software on a K2 summit direct electron detector (Gatan) installed on a FEI Titan Krios (Astbury BioStructure Laboratory, University of Leeds) operating at 300 kV with a Quantum post-column energy filter (Gatan), operated in zero-loss imaging mode with a 20-eV energy-selecting slit. A defocus range of 0.5 to 2.5 μm and a calibrated final sampling of 1.37 Å/pixel was used with the K2 operating in counting mode at 6 e/pixel per second. The total exposure was 48 e2 over 30 frames (1.6 e2 per frame). Movie frames were aligned using MotionCor2 (48) with a patch size of 5 to generate full-dose and dose-weighted sums. Full-dose sums were used for contrast transfer function (CTF) determination in gCTF (49), and then, dose-weighted sums were used in particle picking, processing, and generation of the final reconstructions. Particle processing was performed with helical methods in RELION v2.1.0 (50), using a custom pipeline and scripts to account for microtubule pseudo-helical symmetry. Briefly, microtubules were boxed manually in RELION with a box separation distance of 82 Å (roughly the microtubule dimer repeat distance). The protofilament number of all segments within each microtubule was assigned according to the modal class of those segments after one iteration of 3D classification to 12-Å low-pass–filtered references of undecorated 11- to 16-protofilament microtubules built from multiple asymmetric units of the GMPCPP microtubule atomic model [Protein Data Bank (PDB) ID: 6DPU (51)]. Once the protofilament number of each microtubule had been established, the main 13-protofilament microtubule class was selected for further processing. Rough alignment parameters of each microtubule to its corresponding 7-Å low-pass–filtered 13-protofilament reference were assigned. On the basis of φ angles determined for each segment, median φ angles were assigned to all segments in a given microtubule. Assigned φ angles for each microtubule were checked by 3D classification against 7-Å low-pass–filtered undecorated 13-protofilament microtubule references rotated and translated to represent all possible seam positions and αβ-tubulin registers. For all references used in this step, pixel values were doubled for atoms within the S9-S10 and H1-S2 loops, being the main distinctive features between α- and β-tubulin. Rough final φ angles were assigned according to the modal 3D class of all segments within each microtubule. Finer local refinement was then performed, with or without applied helical symmetry. Final displayed reconstructions were sharpened to local resolutions as determined in RELION, unless stated. Four times binned data were used for the protofilament number assignment 3D classification, unbinned data for the final local refinements, and two times binned data for all other steps. The atomic models of six dimers of undecorated GMPCPP MTs (PDB: 6DPU) with incorporated Taxol [taken from PDB: 5SYF (52)] were fitted into symmetrized density and used as starting models for iterative rounds of model building in Coot (53) and Phenix (54). Extra density contributed by EML4-NTD had low resolution, presumably due to the flexible nature of the MT-EML4-NTD interaction; therefore, EML4-NTD was not modeled into density.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.