Electron micrographs of negatively stained filaments. (a) F-actin-troponin-tropomyosin, (b) F-actin-tropomyosin (b). Note that the presence of troponin (arrowheads), distributed with a 40 nm periodicity in (a) increases the maximum width of thin filaments; also note obliquely oriented tropomyosin strands in (a) and (b) (arrows). Filaments are shown with their pointed ends facing up; polarity was determined by alignment tools in reference36. Scale bar = 50 nm.
Preparation of proteins: F-actin, bovine cardiac troponin and tropomyosin were purified as previously37. SDS-PAGE indicated that the cardiac tropomyosin consisted of greater than 90% aa-isoform and the remainder αβ-isoform; it was presumed to be 20 to 30 percent phosphorylated, as is the tropomyosin in adult cardiac tissue of other mammals38,39. The cardiac troponin as isolated was monophosphorylated and not biphosphorylated as occurs after adrenergic stimulation.
EM and image processing: We have previously shown that a moderate excess of troponin-tropomyosin relative to actin is needed to saturate filaments at the low protein actin concentrations required for successful EM work25,26. Filaments were thus prepared by mixing a two-fold molar excess of tropomyosin or troponin–tropomyosin (40 μM) with F-actin (20 μM) to optimize binding, in 100mM NaCl, 3mM MgCl₂, 1mM NaN₃, 0.2mM EGTA, 1mM dithiothreitol, 5mM sodium phosphate/5mM Pipes buffer (pH 7.0) at 25°C25,26. The mixture was diluted 20-fold, applied to carbon-coated grids and negatively stained with 1% uranyl acetate25,26. Filaments from two preparations of F-actin-troponin-tropomyosin and three preparations of F-actin-tropomyosin were recorded and processed in these studies. EM was done on a Philips CM120 EM at a magnification of X60,000 under low dose conditions (~12 e⁻/Å). Helical reconstruction36 was performed by standard methods as previously25,26. Helical reconstruction was particularly well suited to identify the relatively low density troponin signal observed, as the method takes advantage of layer line indexing information to filter out noise, which otherwise would have obscured the troponin density observed. In addition, robust statistical programs accompany the helical reconstruction package, allowing the significance of weak densities to be assessed by Student’s t-test methodology40,41. Real space reconstruction methods42, treating filament segments as single particles, were also used to confirm results obtained by helical reconstruction and to attempt to sort filament segments according to their tropomyosin positional modes, as previously26.

Statistical significance of densities contributing to reconstructions of Ca²⁺-treated F-actin-troponin-tropomyosin. (a) z-section of reconstruction shown in figure 2d (gold). Note actin subdomains (numbered) and tropomyosin positions (indicated by arrows and labeled Tm) on the inner domain of actin over subdomains 3 and 4. Note also the extra density emerging from subdomain 1 and connecting to tropomyosin (indicated by open arrowhead; the outer edge of the extra density is highlighted in cyan). (b) Densities associated with the z-section in (a) that have a confidence level greater than 99 % (pink). (c) The z-section in (a) superimposed on the statistical map in (b), showing that all densities in (a), including the extra density attributable to troponin, have a high level of statistical significance.

Comparison of cardiac and smooth muscle tropomyosin positions on actin. z-sections of reconstructions of F-actin combined with cardiac muscle tropomyosin (a) and aortic smooth muscle tropomyosin (b). Note the closer association of the smooth muscle tropomyosin density with the actin outer domain (compare white arrows in (a) and (b)). (c) Maps plotting the variance associated with the contributing density points40,41 in the reconstruction in (a) show that a high variance (red) occurs over the site where tropomyosin normally makes contact with actin in the B-state. (d) In contrast, corresponding variance maps of actin – smooth muscle tropomyosin reconstructions show low variance at this site and only comparably high variance (red) at the very edges of the tropomyosin density.

Surface views of thin filament reconstructions showing the position of the high Ca²⁺ troponin extension. Reconstructions of: (a) F-actin (cyan, subdomains numbered on one actin subunit) and (b) F-actin-tropomyosin (green) controls, (c, d, e) F-actin-troponin-tropomyosin: (c, d) high Ca²⁺ (gold), (e) low Ca²⁺ (maroon). Note tropomyosin positions indicated by arrows. High Ca²⁺ reconstructions displayed at two density cutoff thresholds ((c) 5 σ, (d) 2.5 σ greater than the mean density). Note the presence of extra densities in (d) emerging from the centers of subdomain 1, which traverse the face of the domain to abut C-state tropomyosin (open arrowheads). Note also that these densities are absent from F-actin-tropomyosin controls (b) and low Ca²⁺ F-actin-troponin-tropomyosin (e) (each displayed at 2.5 σ). (f) Reconstructions in (d) and (e) superimposed for comparison. Note that the respective tropomyosin strands occupy different positions, with tropomyosin from the high and low Ca²⁺ maps in C- and B-states. Also note that Ca²⁺ specific extra density attributed to troponin (open arrows) crosses the B-state tropomyosin position. All of the reconstructions of thin filaments obtained were aligned to each other and are shown with filament pointed ends facing up. Each map shows densities with amplitudes that are at a threshold of 2.5 sigma over the mean density (with the exception of the map in (c)).

Reconstructions of troponin-free actin – tropomyosin. (a) z-section of reconstruction shown in figure 2b of cardiac tropomyosin bound to F-actin (no troponin). Note that tropomyosin (arrows) occupies a position mid-way between the inner and outer domains of actin, i.e. between actin subdomains 1 and 3 on one side of the filament and between subdomains 2 and 4 on the other side. (b) Tropomyosin densities determined by subtracting densities in the F-actin reconstruction from those of the map in (a) and then superimposing them on the corresponding z-section of F-actin in order to highlight the position of tropomyosin. (c, d) the filaments contributing to the average density map in (a) were sorted according to their best alignment to B- or C-state models. (c) Reconstruction of the sorted filaments that were in the C-state mode; note tropomyosin densities on the actin inner domain (i.e. associated with actin subdomains 3 and 4). (d) Reconstruction of the sorted filaments that were in the B-state mode, with tropomyosin densities on the outer domain (i.e. associated with subdomains 1 and 2). Comparable results were obtained when the skeletal muscle isoform of tropomyosin was examined on troponin-free F-actin, viz. an average position midway between B- and C-state, but here individual filament reconstructions showed a slightly greater bias for the C-state mode than with a found will cardiac tropomyosin.

Cartoon representation of the organization of the thin filament at high Ca²⁺. As previously31, the troponin core domain complexes on either side of F-actin are depicted as W-shaped TnIT structures supporting dumbbell-shaped TnC18,19; actin, grey; tropomyosin, salmon; TnI, cyan; TnC, red; TnT, yellow. At high Ca²⁺, cTerm-TnI dissociates from actin and binds to the N-terminal lobe of TnC. We argue that this releases chemomechanical constraints on troponin and tropomyosin. Troponin then unfurls, here depicted for simplicity as TnT propelling tropomyosin to the C-state position. Other related depictions of troponin on thin filaments can be found in references18,19,43.