Structure of the Flight Muscle Thick Filament from the Bumble Bee, Bombus ignitus, at 6 Å Resolution

Figures

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  Figure 1. Bombus thick filament reconstruction. (A) An electron micrograph of a thick filament. The longitudinal lines in the thick filament backbone are produced by the coiled-coil domains of myosin and paramyosin. The disordered dark transverse densities marked by black arrows are possibly the myosin heads. Occasionally, transverse densities (arrowheads) are observed, which are reminiscent of the Free Heads in Lethocerus thick filaments [8]. (B) The reconstruction shows three full crowns of the Bombus thick filament. The “floating” densities (arrowheads) are the average positions of the myosin heads when helical and 4-fold symmetry are enforced. Non-myosin density, flightin (red) is visible extending from the backbone surface. (C) Transverse view through one crown showing the relative positions of flightin (red), myofilin (yellow), putative paramyosin (purple) and the curved layers (dark gray, light gray and white). Note that both flightin and myofilin have folded domains on the inside surface of the annulus of myosin tails that could contact paramyosin. (D) The 12-crown extended filament, with one curved layer highlighted in blue. The floating densities are not connected to the myosin…

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  Figure 2. Skip residues and proximal S2. (A) Skip residue accommodation regions of Bombus ignitus (grey map envelope) are well aligned with the Lethocerus indicus atomic model (PDB–7KOG) shown in yellow with skip residue colored in purple for the accommodation region and white elsewhere [21], except for Skip 1 where the Lethocerus atomic model falls outside the Bombus envelope due to the latter’s different azimuthal rotation when compared with Lethocerus (white). (B) Superposition of curved layers from Lethocerus (white) and Bombus (blue). When the backbone is low pass filtered to the same resolution that reveals the average myosin head position additional proximal S2 is revealed up to the arrowhead. The Lethocerus proximal S2 (white) prominently bends away from the trajectory of the coiled coils within the backbone as a consequence of Free Head binding to the filament backbone. In Bombus, the proximal S2 (blue) follows the trajectory of the backbone-embedded tail downwards at least as far as the resolved density shown. The Lethocerus density map has been low pass filtered to 7 Å resolution to make it comparable to the displayed Bombus map.

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  Figure 3. Disordered myosin heads of Bombus compared with those of Lethocerus. The Bombus density map (blue) is superimposed on the Lethocerus density map (grey) in both side view (A) and top view (B). (C) An atomic model of the IHM (Blocked Head heavy chain, red; Essential Lght Chain (ELC), blue; Regulatory Light Chain (RLC), yellow; Free Head heavy chain, purple; ELC, green; RLC, orange) along with the myosin head density of the Lethocerus reconstruction is shown. The floating densities from Bombus (blue) represent the average position of otherwise highly disordered myosin heads. The crystal structure of cardiac S2 (red) is aligned to the Bombus S2 density. The S2 atomic model points directly at the floating myosin head density. Note that the Lethocerus features (map and atomic model) are aligned to the cardiac S2 atomic model and not to their position in the relaxed Lethocerus thick filaments. (D) Axial view with atomic models of the Free Head (left) and IHM (right) from relaxed Lethocerus thick filament superimposed on the Bombus density map with the N-terminal domain of the RLC under the proximal S2. The position of the proximal S2 (black arrowheads) is juxtaposed with the inner edge…

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  Figure 4. Multiple sequence alignment of flightin and myofilin of Drosophila, Lethocerus and Bombus flight muscle. (A) The alignment of flightin shows the highly conserved “WYR” motif, which probably corresponds to the common globular flightin density seen in 3-D image reconstructions of thick filaments from the three species. Comparatively, the N- and C- termini are less conserved, although some improvement in conservation is seen at the C-terminus. Among all 112 flightin sequences available in Uniprot, both predicted and observed experimentally, 6 out of 32 C-terminal residues have over 60% similarity (indicated as blue dots below), and if the entries are narrowed down to endopteryogota taxonomy, a total of 80 flightin sequences, 11 out of 32 have high similarity of over 60% (indicated as purple dots below). (B) For myofilin, the N-terminal sequence is well conserved, as is the shape and position of the folded domain seen among the three species, which suggests that they represent the same entity. Conservation decreases after the N-terminal domain, improves somewhat after about 30 residues in Bombus, and becomes very poor toward the C-terminus suggesting that the highly variable parts of…

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  Figure 5. Three non-myosin densities in Bombus thick filament. (A) Bombus flightin molecules (red, solid) are similar among all three species with a small folded domain (labeled 1) in the middle corresponding to the WYR domain [28], a partially ordered extension outside of the filament backbone (labelled 2) on one side of the WYR domain and an extension on the other side (labelled 3). At the N-terminus (labelled 2) Lethocerus (red, mesh) shows the largest extension outside the filament backbone, stabilized by contacts to its proximal S2 [8]. Drosophila has the same feature but with a shorter extension [13] corresponding to only the overlap between Lethocerus and Bombus. The flightin N-terminal extension seen in Bombus leaves the backbone density at a different angle (solid red surface). The flightin C-terminus (labelled 3) overlaps a density (blue, mesh) from a Lethocerus thick filament reconstruction. (B) Bombus myofilin (yellow, solid) shows a novel structure, with two densities, that overlap and contact the Lethocerus myofilin density (yellow, mesh). (C) The Bombus paramyosin (purple, solid) in the central core. (D) The distribution of all three non-myosin proteins in the context of the…

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  Figure 6. AlphaFold2 modeling of Bombus myofilin and flightin. (A) The AlphaFold2 atomic model of the flightin WYR motif fits well into the globular domain in a zoomed-in view (right) that roughly extends from residue 65 to residue 110, which is also the region of conserved sequence and similar reconstruction density in Drosophila and Lethocerus. The full atomic model starts from residue 41 to the residue 151 at the C-terminus (left). (B) The AlphaFold2 atomic model of myofilin only fits well at the N-terminal LKG domain up to residue 36 (right), which corresponds to the region of high sequence and density conservation in Drosophila and Lethocerus, while the rest of the AlphaFold2 model has poor reliability (left).