Computational protocol: Kar3Vik1 Uses a Minus-End Directed Powerstroke for Movement along Microtubules

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Protocol publication

[…] Maleimide-Nanogold® (Nanoprobes, Yaphank, NY) was added in a 2-fold molar excess to 3 nmol of GCN4-Kar3CFVik1C536C, and Nanogold® labeling of the single cysteine residue on Vik1 was allowed to proceed for 10 hours at 4 °C. After labeling, the GCN4-Kar3CFVik1C536C-Nanogold® mixture was applied to a Sephadex-G75 size exclusion column (GE Healthcare, Uppsala, Sweden) to separate the Nanogold®-labeled GCN4-Kar3CFVik1C536C from unbound Nanogold®. Nanogold®-labeled GCN4-Kar3CFVik1C536C was eluted from the column in PME buffer (10 mM PIPES pH 6.9, 5 mM MgCl2, 1 mM EGTA) with 100 mM KCl, 0.5 mM TCEP and 0.1 mM ATP. Labeling efficiency was shown to be 70–80% determined by separating Nanogold®-labeled Vik1C536C from unlabeled Vik1C536C by SDS-PAGE and comparing their relative band intensities on the gel using ImageJ (National Institutes of Health, Bethesda, MD). [...] Cryo-EM CCD images were visually inspected for fully decorated MTs with fifteen protofilaments. Using IMOD , fifteen-protofilament/two-start helical MTs were extracted from the images and rotated to lie horizontally. Helical processing was carried out using PHOELIX as described previously . In brief, each MT was computationally straightened and layer-line information was extracted. Layer-line data from a large number of MTs were shifted to a common phase origin using a reference and subsequently averaged. Three rounds of averaging were carried out with iterative improvement of the reference used to align the data. Helical averaging was always completed with more than one reference to ensure that final averages were not biased by the reference used. Only datasets with a phase residual of less than 20 relative to the reference were included in the final reconstruction. The final dataset was truncated to a maximum resolution of 2.2 nm which, at the imaging conditions used here marks the position of the first so-called Thon ring produced by the contrast transfer function (CTF: see also ). 3-D density maps of the averages were visualized in IMOD. Surface rendering of the maps was carried out in UCSF Chimera . Phase-amplitude plots of the merged average () and the overlay of all individual datasets () provide an objective measure of the data quality. The resolution of the maps is estimated by Fourier-Shell correlation to be around 2.2–2.5 nm ().The number of individual datasets and approximate number of asymmetric units included in each of the helical reconstructions is shown in . […]

Pipeline specifications

Software tools ImageJ, IMOD, PHOELIX, UCSF Chimera
Applications cryo-EM, Microscopic phenotype analysis
Diseases Movement Disorders
Chemicals Adenosine Diphosphate, Adenosine Triphosphate, Nucleotides