TOWARDS ATOMIC INTERPRETATION OF F-ACTIN FILAMENT 3-DIMENSIONAL RECONSTRUCTIONS

We have recorded dark field images of negatively stained F-actin filam ents polymerized with 2 mM MgCl2 and 50 mM KCl with a scanning transmi ssion electron microscope and computed 3-D reconstructions using a hel ical parameter search to optimize simultaneously the helical repeat le ngth, the radial position of the filament axis, and the helical select ion rule. The resulting optimized averaged filament 3-D reconstruction at 2.5 nm resolution is remarkably similar to an atomic model of the F-actin filament. By comparison, several structural features of the re construction can be interpreted at the level of distinct secondary str ucture elements, and predictions made by the atomic model could be ver ified: for instance, the density connecting the two long-pitch helical strands in our reconstruction co-localizes with an extended beta-hair pin, the ''hydrophobic loop'' (i.e. residues 262 to 274), which accord ing to the atomic model establishes the major intersubunit contact bet ween the two long-pitch helical strands. The most pronounced structura l variations among individual filament 3-D reconstructions were observ ed in (1) the details of the intersubunit contact pattern between the two long-pitch helical strands, and (2) the exact size and shape of su bdomain 2 of the F-actin molecule, which appears rather flexible and e asily deformed. In addition, we found that all phenotypes of F-actin f ilament 3-D reconstructions that arise from small deviations from the optimal helical parameters or from lowering the nominal resolution exh ibited stronger intersubunit contacts between than along the two long- pitch helical strands, a structural feature that has been emphasized f or a number of F-actin filament 3-D reconstructions in the past. Since this is clearly at variance with the relative strength of the intersu bunit contacts as predicted by the atomic model, it may represent an a rtifactual structural feature arising from low-resolution data or subo ptimal helical data processing, and should therefore be interpreted wi th caution in terms of indicating chemical, mechanical or conformation al states of the F-actin filament.