The 3D structure of rabbit skeletal muscle F-actin filaments was determined through the use of electron cryo-microscopy and existing image processing procedures (Hanein et al., 1997). The alignment of the atomic model of F-actin (Lorenz et al., 1993) to the pre-aligned density maps was done manually using the program O (Jones et al., 1991). As seen the envelope encases the alpha-carbon backbone of actin. In all the presentations the pointed end of the actin filament is up. The F-actin diameter is approximately 100Å. Low-dose images of frozen-hydrated preparations were recorded using 120kV electrons, at a nominal magnification of 60,000x, and ~2.2µm defocus (electron dose ~10e-/ Ų). Selected electron micrographs were digitized with an Eikonix 1412 densitometer at a scanning raster of 25µm (~4.3 Å/pixel). Image processing and three dimensional reconstruction procedures employed the Brandeis Helical Package (Owen et al, 1996). Rabbit skeletal muscle actin was prepared according to Pardee and Spudich (Pardee and Spudich, 1982), flash frozen in liquid nitrogen, and stored as 1mg aliquots at -80 -degrees- C. Prior to use, aliquots were thawed, dialyzed against G-actin buffer (2mM Tris pH 8.0, 0.2mM CaCl₂, 0.2mM ATP, and 1mM DTT), and clarified at 70K rpm for 30 minutes in a Beckman TLA-100 rotor. The actin was polymerized by addition of NaCl to 20mM and MgCl₂ to 2mM for 30 minutes at room temperature and stored on ice

More details can be obtained in Hanein et al., 1997. [abstract] [full text]