Figure 1.

Surface views of one 360° helical repeat (~720Å) of rigor acto-CSk S1 (A) acto-wt Dros S1 (B), acto-IFI-EC S1 (C), and acto-Leth (D) density maps. The acto-S1 complexes share similar features including identical actin-motor domain positions and a long (~85 Å) LCD projecting away from the actin filament, labeled in panel A: MD - motor domain, ELC- essential light chain region, RLC-regulatory light chain region. Protein Purification. Skeletal myosin was prepared from chicken pectoralis muscle as previously described ³⁶. Myosin from insect IFM was purified from either half of a single Leth thorax or ~ 300 wt Dros or 450–500 IFI-EC thoraces (per myosin preparation) as previously described ¹⁸. The wt Dros myosin yield was ~ 3.5 µg/fly where as the routine myosin yield from one Leth half thorax was ~ 500 µg. F-actin was prepared from rabbit muscle as previously described ³⁷. S1 Preparation. The papain-Mg²⁺ myosin chicken S1 fragment was prepared as previously described ³⁸ with the following modifications. Briefly, myosin at 5 – 20 mg/ml was suspended in 0.2 M ammonium acetate (pH 7.0), 2 mM MgCl₂, and 1 mM DTT. The protein was digested with 0.03 mg papain (Worthington Biochemicals, Lakewood, NJ-activated at 1 mg/ml as per manufacturers instructions) per ml of myosin solution for 8 min. at 25°C. The reaction was stopped with 3 mM iodoacetic acid with one mini protease inhibitor tablet added/10ml solution (Roche Biochem, Mannheim, Germany) and placed on ice. The suspension was subjected to ultra-centrifugation (218 K × g) for 30 min. at 4°C to pellet the undigested myosin and rod while the soluble S1 fragment remained in solution. Insect S1 was prepared in a similar manner with the following exceptions: whole myosin was resuspended at 2 – 4 mg/ml in 2 mM KCl, 3 mM EDTA, pH 7.0, 20 mM potassium phosphate, pH 6.7, 5 mM β-mercaptoethanol and 5 mM MgCl₂ and digested with papain at 1:1000 w/w, enzyme to total protein, activated as described above, for 14–17 min. at 25°C. Longer digestion times yielded more S1 but resulted in significant digestion of the regulatory light chain. Concentration was determined using the E^1%₂₈₀ = 7.5, molecular weight of ~90 KD ³⁹. S1 purity was assessed by SDS-PAGE. Acto-S1 complex preparation. Rabbit F-actin (0.05 mg/ml) was decorated with rigor (nucleotide free) S1 on holey copper carbon-coated grids (400 mesh, Ted Pella, Inc., Redding, CA, or 400 mesh Quantifoil, Structure Probe, Inc., West Chester, PA). Specimens were frozen in liquid ethane and stored in liquid nitrogen. Cryo-electron microscopy and image analysis. Specimens were examined with a Philips CM200 FEG (120 kV, 36K × magnification, −178°C). Images were acquired at 1.5–1.8 .m under focus using low dose conditions on Kodak SO-163 film. Fully decorated filaments were scanned (Perkin Elmer 1010 scanner) and subjected to helical image analysis using Phoelix ⁴⁰. See Table 1 for a summary of the data and image processing results. S1 modeling was done manually using the program O ⁴¹ and the figures were created with AVS (Advanced Visual Systems, Inc., Waltham, MA).