EVALUATION OF HIGH-RESOLUTION SHADOWING APPLIED TO FREEZE-FRACTURED, DEEP-ETCHED PARTICLES - 3-D HELICAL RECONSTRUCTION OF SHADOWED ACTIN-FILAMENTS

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 shadowin g must vary systematically from subunit to subunit, the computed trans forms of isolated filaments were characteristic of the helical actin t ransform. Helical reconstruction was therefore judged to be valid. The theoretical basis for such reconstruction is outlined. The reconstruc tions showed an average thin (about 3 nm) layer of shadow on the filam ent surface and both the outer and the inner surfaces of the shadow la yer could be visualized. By comparison with the F-actin structure post ulated 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 ''shadowe d'' filaments can thus be correlated axially and azimuthally with know n actin structures and, in principle, features such as myosin head loc ation on decorated filaments can be determined. The result emphasizes the amount of detail present in good quality images of shadowed partic les and, in this case, shows that detailed evaluation of molecules lab eling actin can be made. (C) 1994 Academic Press, Inc.