Taxol-stabilized microtubules were decorated with Ndc80 bonsai after application to glow-discharged C-flat grids (Protochips), then plunge-frozen in ethane slush. Images were collected on KODAK SO-163 film with a Tecnai F20 electron microscope operating at 200kV at a nominal magnification of 50,000× with dose of 15 electrons/Å2. Micrographs were digitized with a Nikon Super CoolScan 8000 scanner with a step size of 6.35 μm. Image processing and projection-matching alignment was carried out using programs from the EMAN, IMAGIC, and SPIDER packages, and final refinement and CTF correction was performed with a version of FREALIGN adapted for this work to implement helical symmetry. Visualization and molecular docking was performed with UCSF Chimera. Amplitude-weighted difference maps were calculated using the program DIFFMAP (available at: http://emlab.rose2.brandeis.edu/software ). References for all image analysis and visualization software can be found in the Methods section.
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Cryogenic Imaging of Ndc80-Stabilized Microtubules
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Other organizations : University of California, Berkeley, European Institute of Oncology, Lawrence Berkeley National Laboratory, Howard Hughes Medical Institute, Brandeis University, Italian Institute of Technology
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Variable analysis
independent variables
- Application of Ndc80 bonsai to glow-discharged C-flat grids
- Plunge-freezing in ethane slush
- Microtubule decoration with Ndc80 bonsai
- Electron microscope imaging and micrograph digitization
- Taxol-stabilized microtubules
- Electron microscope operating at 200kV with a nominal magnification of 50,000× and a dose of 15 electrons/Å^2
- Micrograph digitization with a Nikon Super CoolScan 8000 scanner at a step size of 6.35 μm
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