Computational protocol: Structure and Dynamics of Single-isoform Recombinant Neuronal Human Tubulin*♦

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Protocol publication

[…] Individual ∼2.2 e−/Å2 frames were globally aligned using IMOD scripts () then locally aligned using the Optical Flow approach () implemented in Xmipp (). The full dose of ∼50 e−/Å2 was used for particle picking and CTF determination in CTFFind3 (), whereas ∼25 e−/Å2 was used in particle processing to center particles and determine their Euler angles. Euler angles and shifts determined using ∼25 e−/Å2 dose were used to generate reconstructions from either the first ∼25 or ∼12 e−/Å2 of the exposure. Kinesin-3 microtubules were manually boxed in Eman Boxer (), serving as input for a set of custom-designed semi-automated single-particle processing scripts utilizing Spider and Frealign as described previously () with minor modifications. 10,164 particles or 142,296 asymmetric units were used in the final reconstruction, which was assessed for overfitting using a high resolution noise-substitution test (). Using local resolution estimates determined with the blocres program in Bsoft, the reconstruction was sharpened with a B factor of −180 up to a resolution of 5.5 or 4 Å for visualization of kinesin or tubulin densities, respectively. The overall resolution of the reconstruction is 4.2 Å (FSCtrue, 0.143 criteria) () encompassing a resolution range of ∼3.5–5.5 Å. The best regions of the reconstruction are within the tubulin portion of the complex (B and ) from which we built an α1A/βIII microtubule model. The quality of our reconstruction was sufficient to confirm that GMPCPP was found in the E-site (C) and GTP in the N-site. [...] The polypeptide model of the unmodified α1A/βIII-tubulin GMPCPP microtubule was built directly into density in Coot () using PDB 3JAT () as a starting model. The structure was refined under symmetry restraints in REFMAC version 5.8 (). Secondary structure and reference restraints based on the high resolution tubulin crystal structure PDB 4DRX () were generated with ProSMART (). Model building in Coot and refinement in REFMAC were repeated iteratively until the quality of the model and fit were optimized (supplemental Table 1). […]

Pipeline specifications

Software tools IMOD, Xmipp, CTFFIND, EMAN, Frealign, Bsoft, Coot
Applications cryo-EM, Protein structure analysis
Organisms Homo sapiens