Article Figures & Data
Figures
- Figure 1. Myosin filament features.
(A) Diagram of a myosin molecule with two equivalent heads and an α-helical coiled-coil tail. Proteolysis at two sites (arrowheads) fragments the molecule into two separate heads (S1) and two tail segments (S2 and LMM [light meromyosin]). (A, B, C) Vertical line represents 1,000 Å in panel (A) and 100 Å in (B, C). (B) The interacting heads motif (IHM). In the IHM, the two heads are not equivalent. Instead, the actin-binding domain of one head (blocked) contacts the adjacent head (free) whose actin-binding domain is not blocked. The inset shows the space-filling structure of PDB 1I84 (Wendt et al, 2001). In filaments, the free head is usually juxtaposed to the thick filament backbone effectively preventing it from binding actin in the relaxed state. (C) The IHM placed within the Lethocerus thick filament reconstruction at 20 Å resolution. The black disk approximates the orientation of a best plane drawn through the IHM. This orientation is unique in striated muscle. (D) Schematic diagram showing the relative placement of the giant proteins, kettin, projectin, obscurin, and stretchin-klp within a sarcomere. Projectin binds mostly at the filament tip, obscurin to the M-band (bare zone), and kettin to the thin filament and projectin. Stretchin-klp binds along the main shaft of the thick filament but not in the bare zone or at the filament tips. (A, B, C) Coloring scheme in (A, B, C)—blocked head: heavy chain, red; essential light chain, blue; regulatory light chain, yellow; free head: heavy chain, purple; essential light chain, green; regulatory light chain, orange. Scale bar in (A) is 1,000 Å, in (B, C), 100 Å. (D) Coloring scheme for (D)—Fn3 domains, light green; Ig domains, blue; Ig domains of stretchin-klp, pink; kettin, red; obscurin kinase domains, yellow. (D) Adapted from Bullard et al (2005). (A, B, C) from Hu et al (2016).
- Figure 2. Typical thick filament images and reconstructions of WT and mutant flies.
(A) WT-isolated thick filaments. In the adjacent A-bands, numerous densities (myosin heads) project from the surface. (B) Thick filament from regulatory light chain mutant flies. Images for this set recorded using a Volta phase plate. Note that the density across the diameter of the bare zone is uniform but across the A-band is lighter in the middle, suggesting the filaments are hollow. (C) Image of the WT thick filament showing the disorder of the myosin heads and the smoothness of the bare zone, outlined in the white box. (D) WT reconstruction after imposing helical symmetry and application of local deblur. (E) Mutant reconstruction after similar treatment. (D, E) Images in panels (D, E) low-pass filtered to 7 Å resolution for the backbone and 40 Å resolution to display the floating head densities.
- Figure S1. Filament segment selection.
(A) Filaments are picked from the M-band (crown 0), but not all of them have the same length. Some of the filaments end outside of the image and others go over the carbon grid. Note that the disordered heads are clearly visible in the A-band region, but the bare zone is very smooth. (B) Distribution of the tube length shows most segments used in the reconstruction come from an area close to the M-band. The distribution does not change after classification showing that distance from the M-band is not a major factor. Drosophila obscurin, the one protein whose visibility might be affected by a paucity of filament segments near the bare zone, is only long enough to reach the first crown of the A-band. The number of filament segments beyond 90 crowns is too small a fraction of the total for projectin to appear in the reconstruction.
- Figure S2. Resolution determination for the W1118 thick filament reconstruction.
Resolution was computed using the version of Mono Res embedded within Scipion (Gomez-Blanco et al, 2018). The color bars show the local resolution on each surface in Ångstroms. (A) Side view of local resolution map of the wild-type Drosophila. Map used was at 2.5 threshold which is the optimal threshold that includes most of the features with various local resolutions. Most of the map is at ∼7.5 Å resolution. (B) Slices of the resolution map from in the Z direction. (C) FSC plot of the map generated from two half maps using Scipion.
- Figure S3. Resolution determination for the Drosophila Dmlc2[Δ2-46; S66A,S67A] mutant reconstruction.
The color bar shows the local resolution on the surface. (A) Side view. Map used was at 0.8 threshold which is the optimal threshold that includes most of the features with various local resolutions. Most of the map is at ∼8 Å resolution. (B) FSC plot of the map generated from two half maps using Scipion.
- Figure S4. Reconstructions obtained using alternate symmetries.
The reconstruction imposing C3 symmetry resulted in three rather massive densities with slim connections between them, which is inconsistent with observation from electron microscopy of transverse sections through striated muscle (Reedy et al, 2000; Farman et al, 2009), which generally show relatively uniform density for the filament backbone. The reconstruction done with C2 symmetry has more uniform density within which the myosin tails are arranged.
- Figure 3. Arrangement of myosin and non-myosin proteins.
(A) Four symmetrically placed myosin tails (blue) segmented from a reconstruction extended 12 crowns. As in Lethocerus thick filaments, myosin tails run mostly parallel to the filament axis with a slight tilt inward toward the C terminus. (B) View looking down the filament axis showing the “curved molecular crystalline layers” (ribbons) (Squire, 1973). 10 myosin tails in each of the four asymmetric units, arising from the fourfold symmetry, are numbered sequentially according to their 145 Å axial offsets. Each ribbon consists of myosin tails offset axially by 3 × 145 Å, that is, 1, 4, 7, and 10; 2, 5, and 8; and 3, 6, and 9, starting from the point where the tail enters the backbone. Ribbons are colored white, light, and dark gray. (C) Longitudinal view from the outside. Non-myosin proteins flightin (red) and myofilin (yellow) are embedded among the myosin tails. A third, possibly stretchin-klp (purple and pink) is on the outer surface. (D) Myosin tails of Drosophila (gray) and Lethocerus (pink mesh) superimposed as a ribbon. With the exception of the proximal S2 of Lethocerus, which bends to the left, the same feature in Drosophila is mostly disordered but what is visible appears to follow a straight trajectory. Note that the density threshold for the Lethocerus reconstruction was chosen to be the minimum that would show the position of the free head without blurring the proximal S2.
- Figure 4. Non-myosin proteins of the Drosophila thick filament reconstruction.
(A) View from the outside showing the myosin head density in relationship to the non-myosin proteins. (B) View from the outside at higher magnification looking through the myosin tails. The putative stretchin-klp densities (purple and pink) of Drosophila, which are not seen in Lethocerus, are found only on the outside of the thick filament backbone. (C) View from the inside looking out. Flightin (red) extends through the gray ribbon, whereas myofilin (yellow) is at the edge of the ribbon and the blue protein positioned on its surface. (D) The blue density binds the inner surface of the gray ribbon; the yellow (myofilin) density binds between the gray and white ribbon; the red density penetrates the gray ribbon. (E) Red, blue, and yellow non-myosin densities of Drosophila superimposed on the corresponding feature of Lethocerus displayed as mesh. (F) View from the outside showing the putative stretchin-klp density. An atomic model of an I-set domain has been built into two of the densities. The third (pink) is shown without an atomic model. Threshold for I-set/tail is 4.65 and threshold for stretchin linker is 1.00. (G) The paired Ig-like densities shown on a ribbon model of the myosin tail. (H) The putative stretchin-klp densities superimposed on a Lethocerus thick filament reconstruction where they appear to pass under the free head and S2. Note that the density threshold for the Lethocerus reconstruction was chosen to be the minimum that would show the position of the free head without blurring the proximal S2. Coloring scheme same as Fig 3. One ribbon is colored gray. (A, B, D) Myosin tails in panels (A, B, D) are at 50% transparency.
- Figure S5. Lethocerus thick filament ribbon aligned to the Drosophila thick filament backbone.
Putative stretchin-klp I-set densities are superimposed on a Lethocerus myosin ribbon showing density from the myosin-free head. I-set domains are colored purple, Lethocerus myosin violet, Drosophila thick filament backbone is light gray, and linker densities shown in pink. (A) Side view showing one I-set domain positioned near the proximal S2 of Lethocerus. (B) Front view showing one I-set domain and linker domain (pink) positioned near the free head regulatory light chain. (C) Side view from blocked head toward free head showing closeness of I-set domain to the proximal S2 and the myosin heads.
Tables
Gene Common name Accession no. Molecular weight (kD) Myosin molecules per protein molecule (myofibril) Myosin molecules per protein molecule (filamentsa) Fln Flightin P35554 20,656 1.3 ± 0.4 1.5 ± 0.5 Mf Myofilin Q9VFC7; C1C553 41,667 2.0 ± 0.7 2.0 ± 0.6 strn-mlck Stretchin-mlck A1ZA73 215,065 3.4 ± 1.1 7.9 ± 2.5 Prm Paramyosin P35415 102,338 23.5 ± 7.9 30.5 ± 9.6 Prm Miniparamyosin P35416; M9NDM6 74,277 9.7 ± 3.3 14.7 ± 4.6 Bt Projectin L0MN91 992,527 30 ± 10 Not detected Sls Titin/kettin Q9I7U4-2 548,598 34 ± 12 Not detected unc-89 Obscurin A8DYP0 475,000 42 ± 14 62 ± 20 ↵a Sufficient specimen for only a single measurement. Uncertainties were determined from variability between the relative ratios generated using peptides from the myosin heavy chain, essential, and regulatory light chains.
Lethocerus indicus Drosophila melanogaster Reference Filament length 2.3 μm 3.2 μm Reedy (1967) and Gasek et al (2016) Rotational symmetry C4 C4 Morris et al (1991), Reedy et al (1981), and this work Helical parameters 145 Å, 33.98° 145 Å, 33.86° Hu et al (2016), Irving and Maughan (2000), Perz-Edwards et al (2011), and this work Non-myosin proteins Protein Molecular weight Ratio to Mhc Molecular weight Ratio to Mhc Flightin 19 kD 1:2 20 kD 1:2 Qiu et al (2005) Myofilin 30 kD 1:2 20 kD 1:2 Qiu et al (2005) Paramyosin 107 kD 1:7a 102 kD 1:15 Bullard et al (1973), Vinos et al (1991), Becker et al (1992) Miniparamyosin 62 kD 55 kD Becker et al (1992), Maroto et al (1996) Projectin 800 kD 800–1,000 kD Lakey et al (1990), Ayme-Southgate and Southgate (2006) Kettin 700 kD 540 kD Lakey et al (1993), Bullard et al (2006) Stretchin-klp 225, 231 kD Patel and Saide (2005) ↵a Lethocerus cordofanus and Lethocerus maximus.
- Video 1
Drosophila ribbon (gray) superimposed and aligned with one from Lethocerus, shown in wire mesh. The movie shows the rather high similarity between the Lethocerus and Drosophila ribbons. The Lethocerus density shows the proximal S2 as well as a partial outline of the free head and the blocked head regulatory light chain. The orientation of all movies has the M-line at the top, Z-disk at the bottom.Download video
- Video 2
Longitudinal slabs cutting through the segmented filament backbone. Myosin tails are colored in white, light gray, and dark gray to differentiate the separate myosin ribbons. Flightin is colored red, myofilin in yellow, and the purple and pink densities on the backbone surface are putative strechin-klp. The blue density is from an unknown non-myosin protein, possibly a domain of flightin or myofilin. The orientation of all movies has the M-line at the top and Z-disk at the bottom.Download video
- Video 3
A 360° view of the filament backbone. Flightin is colored in red and purple and pink densities are putative strechin-klp. The proximal S2 connecting tails to myosin heads is not resolved because of the myosin heads being disordered. The orientation has the M-line at the top and Z-disk at the bottom.Download video
- Video 4
The four main non-myosin densities observed in Drosophila flight muscle thick filaments. Flightin is red, myofilin is yellow, putative stretchin-klp purple and pink, and unknown blue. Three ribbons are colored dark gray, light gray, and white. An I-set domain atomic structure (PDB 2YXM) displayed as a blue ribbon diagram has been fit into the putative stretchin-klp densities. The fit is good but not definitive at this resolution. Just before the movie starts, the myosin tail N terminus, that is, the beginning of the proximal S2 is visible in the upper right hand corner. The orientation has the M-line at the top and Z-disk at the bottom.Download video
- Video 5
Putative strechin-klp Ig domains (purple) and long linker (pink) decorating the thick filament backbone. We interpret the pink-colored feature as an average over several long linker structures. Consequently, its shape is possibly meaningless. The three densities appear to define a left-handed helical track, although the linkers between the features are not resolved. This is not a definitive assignment but seems more reasonable as the separation distance between the pink and purple densities is less with this interpretation. The “floating” densities are quite possibly the disordered myosin heads. The orientation of all movies has the M-line at the top and Z-disk at the bottom.Download video
- Video 6
Densities located on the thick filament backbone. At 50% transparency and aligned to the Drosophila backbone is a ribbon from the thick filaments from Lethocerus indicus. The contour cutoff for the Lethocerus ribbon is sufficient to show a partial outline of the free head and the blocked head regulatory light chain. Nevertheless, the Lethocerus myosin heads have enough volume to show close proximity to the stretchin-klp density (pink). One of the stretchin-klp densities also lies in close proximity to the Lethocerus proximal S2 where it leaves the close packing of the filament backbone. The orientation of all movies has the M-line at the top and Z-disk at the bottom.Download video
Supplemental Data 1.
[LSA-2020-00823_Supplemental_Data_1.xlsx]Mass spectrometry myofibril data.
Supplemental Data 2.
[LSA-2020-00823_Supplemental_Data_2.xlsx]Mass spectrometry isolated filament data.
Supplemental Data 3.
[LSA-2020-00823_Supplemental_Data_3.docx]Supplemental methods.
