title: Evaluation of high-resolution shadowing applied to freeze-fractured, deep-etched particles: 3-D helical reconstruction of shadowed actin filaments.
abstract: Images of shadowed F-actin filaments on mica surfaces obtained using a quick-freeze, freeze-fracture, deep-etch technique were subjected to conventional 3-D helical reconstruction methods. Although the shadowing must vary systematically from subunit to subunit, the computed transforms of isolated filaments were characteristic of the helical actin transform. Helical reconstruction was therefore judged to be valid. The theoretical basis for such reconstruction is outlined. The reconstructions showed an average thin (about 3 nm) layer of shadow on the filament surface and both the outer and the inner surfaces of the shadow layer could be visualized. By comparison with the F-actin structure postulated by Holmes et al. (1990) on the basis of the known structure of the actin monomer, it is shown that, at the resolution considered, the inner surface of the shadow provides a reasonably faithful outline of the molecular surface. This, in turn, confirms that the original 3-D structure of the protein molecules has been well preserved throughout the whole preparation procedure up to the final replica. The "shadowed" filaments can thus be correlated axially and azimuthally with known actin structures and, in principle, features such as myosin head location on decorated filaments can be determined. The result emphasizes the amount of detail present in good quality images of shadowed particles and, in this case, shows that detailed evaluation of molecules labeling actin can be made.
authors.affiliation: Biophysics Section, Blackett Laboratory, Imperial College, London, United Kingdom.
authors.forename: E P
authors.initials: EP
authors.lastname: Morris
authors.forename: E
authors.initials: E
authors.lastname: Katayama
authors.forename: J M
authors.initials: JM
authors.lastname: Squire
mesh_terms: D000199:Actins / Q000737:chemistry / Q000648:ultrastructure*; D005613:Freeze Etching / Q000379:methods; D005614:Freeze Fracturing / Q000379:methods; D008854:Microscopy, Electron / Q000379:methods; D008961:Models, Structural; D017433:Protein Structure, Secondary
chemical_list: D000199:Actins