Computational protocol: A novel approach to the structural analysis of partially decorated actin based filaments

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Protocol publication

[…] Thin filament preparation and viewing. Thin filaments were isolated from two sources; goldfish (Carassius auratus) body muscle and mouse cardiac muscle. Muscles were prepared in relaxing solution as described in . Electron micrographs were recorded of negatively-stained filaments applied to a thin carbon film support over the holes of holey carbon grids. The goldfish images were collected on a JEOL 1200EX at a nominal magnification of 20,000× and the mouse cardiac images recorded on an FEI T 12 at a nominal magnification of 42,000× under low dose conditions. The images were digitised on a Nikon Coolscan 8000ED with a step size of 6.35 μm. A 3D reconstruction of the goldfish data has been published in .Image processing. Image processing software packages were used as follows: Imagic () the MRC suite of programs (); Spider () and ImageJ (http://rsb.info.nih.gov/nih-imageJ). The straightening of filament images was achieved using an in-house straightening program and the straighten plugin for ImageJ (). Helical reconstruction was carried out using adapted programs from the MRC Image package.Single particle analysis and imposing screw symmetry. The image processing package Imagic provided the framework for the single particle analysis (). Adaptations to the standard procedures, specific to filamentous particles, were implemented including the modified back-projection algorithm ().Rendering in PyMol. 3D models, electron density maps and PDB structures were rendered using PyMol (www.pymol.org). A hybrid method (using Imagic and PyMol) has been established that allows EM density maps to be displayed in PyMol at a theoretical volume corresponding to the appropriate molecular weight. […]

Pipeline specifications

Software tools IMAGIC, SPIDER, ImageJ, PyMOL
Application Microscopic phenotype analysis