Research ArticleBIOCHEMISTRY

Structure of myosin filaments from relaxed Lethocerus flight muscle by cryo-EM at 6 Å resolution

See allHide authors and affiliations

Science Advances  30 Sep 2016:
Vol. 2, no. 9, e1600058
DOI: 10.1126/sciadv.1600058
  • Fig. 1 Myosin diagrams.

    (A) The classic myosin diagram pictures two equivalent heads and an α-helical coiled-coil rod domain. Proteolysis at two sites (arrowheads) fragments the molecule into two separate heads (S1) and two rod segments [S2 and LMM (light meromyosin)]. (B) Bipolar thick filaments form, with the rods packed into the backbone (gray cylinder) and the heads helically arranged on the surface. During contraction, the heads bind the adjacent actin filaments and pull them toward the central, head-free bare zone. (C) The S1 crystal structure [1DFL (92)] and diagram show the head divided into a bulky motor domain (MD), an Src homology 3 domain (SH3), and the lever arm consisting of the essential and regulatory light chains (ELC and RLC) wrapping around a long central α helix (bracket) that ends in a right-angle hook (arrowhead) (93). (D) In the IHM, the two heads are not equivalent. Instead, the actin-binding domain of one head (blocked) contacts the adjacent head (free). The inset shows the space-filling structure of 1I84 (9). Blocked head: red, myosin heavy chain; blue, ELC; yellow, RLC. Free head: purple, heavy chain; green, ELC; orange, RLC. Scale bars, 100 Å; scales are equivalent in (C) and (D).

  • Fig. 2 Cryo-EM density map at 20 Å resolution.

    (A) An electron micrograph of a thick filament, processed using damage-compensated motion correction (81), shows crowns every 145 Å, where myosin heads project from the filament backbone (lines). (B) Longitudinal view of the 3D reconstruction, with the filament axis vertical and the bare zone toward the top. A single S2 tether is identified by the red conical arrow, which is depicted in the same position but from different viewing directions in (D) to (F). (C) A pseudofilament generated by applying the helical parameters to the reconstruction volume. For context, this volume is shown as a background envelope in subsequent figures. (D) Longitudinal view of PDB 1I84 fit into the map. The coloring scheme is the same as in Fig. 1D. (E) An M-ward axial view shows that the free head makes three contacts with the backbone (arrowheads). (F) A Z-ward axial view, tilted slightly to look down the S2 tether, shows that the lever arm hook points directly to the S2 tether. For clarity, the RLCs have been omitted in (F). Scale bars, 100 Å; scales are equivalent in (D) to (F).

  • Fig. 3 Myosin filament backbone.

    (A) The 5.5 Å map illustrates the myosin rod density within the 20 Å envelope. One myosin rod is colored blue and the free-head RLC densities are orange. (B) A portion of (A) magnified ×2 and rotated 34° shows the unwound coiled coils in the Skip 1 region of the rod (blue). The free-head RLC density (orange) is closely juxtaposed to the S2 of another myosin molecule. (C) An M-ward view of the IHM juxtaposed with myosin rods shows the free-head heavy chain and RLC contacts to the rods. (D) Three symmetry-related molecules in one crown are shown. The arrows on the right mark the coiled-coil crossovers. (E) Z-ward view of the path of a single rod in axial projection, color-coded red to blue by Z-height. Large circle, 100 Å radius. Two 10 Å–diameter circles surrounded by a 20 Å circle are placed where the clipping plane intersects the S2 tether and represent the coiled coil of the rod. (F) Skip region atomic structures fitted to the rod density. Skip residues are colored red; red spheres indicate their predicted positions from the contour length of the rod. Arrowheads indicate bridging densities in the map filled by bulky side chains.

  • Fig. 4 Myosin rod arrangement.

    (A) Rods in one quadrant numbered by their crown level, increasing toward the viewer, M-ward view. Other quadrants are equivalent by fourfold symmetry. Lines mark three groupings of the rods into ribbons, colored blue, pink, and green. There is no indication of a 40 Å subfilament arrangement (inset). (B) A ribbon seen flatwise shows the path of one myosin molecule across the ribbon. (C) In a 3600 Å–long pseudofilament, the helical ribbon turns through 100° and is therefore seen edgewise near the bottom and flatwise near the top. (D) For 20 Å coiled coils, the distance between rod centers, D, is 17 to 20 Å, depending on the angle of contact. In the 14 Å contact on the right, rotating the coiled coils will cause overlap; thus, this type of contact is only possible with unwinding or at the end of one coiled coil. (E to G) The reference, Molecule 0 (gray), is shown with the adjacent molecules contacting it, as well as the distance between them in color-coded graphs. (E) Distance to Molecules +3 and −3 (blue and red, respectively) within a ribbon. (F) Distance to Molecules +1, −2, and −5 (blue, red, and green, respectively) in the adjacent ribbon. (G) Distance to Molecules +5, +2, and −1 (green, red, and blue, respectively) in the other adjacent ribbon.

  • Fig. 5 Nonmyosin densities.

    (A) Nonmyosin densities in the region between crowns are colored red, yellow, blue, and green. (B) A Z-ward axial view of the 5.5 Å map shows the extra densities tightly packed among the rods. (C) This longitudinal view looks from inside the filament, toward the rods from four different ribbons, which are alternately colored light or dark gray. The individual densities are inserted between the rods of a given ribbon but also contact and therefore bridge adjacent ribbons. (D) A single ribbon is shown with the extra densities that contact it. The red density penetrates near the top of a large split in the ribbon (star). (E) A single myosin molecule is shown in orthogonal views with the extra densities that contact it. Numbers represent the predicted amino acid residue of the Drosophila myosin sequence (see text) at 145 Å intervals. Double arrows indicate the predicted location of the four myosin skip residues.

  • Fig. 6 Paramyosin core.

    The paramyosin core (purple) is shown in context, with the rods and extra proteins nearest the viewer removed. Note the axial variations in the density, which appear as a continuous ring of density just below the level of the crowns (solid line) and as axially oriented rods in the regions between crowns (dashed line), where the nonmyosin densities are located.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/9/e1600058/DC1

    table S1. Sequence identity among representative myosins.

    Modeling the paramyosin core

    In-depth discussion of subfilaments versus ribbons

    The IHM and possible relevance to stretch activation

    fig. S1. Resolution of the reconstruction.

    fig. S2. Comparison of the Fourier transform of the map to the x-ray pattern of whole muscles.

    fig. S3. Poor fitting of the IHM with the blocked head contacting the backbone.

    fig. S4. Orientation of the IHM in Lethocerus filaments compared to tarantula filaments.

    fig. S5. Schematic illustration of the paramyosin modeling.

    fig. S6. Model of the paramyosin core.

    fig. S7. Illustration of particle classification scheme.

    fig. S8. Key to movie S3.

    movie S1. Fitting of IHM structure into the 20 Å map.

    movie S2. Myosin molecule within the filament.

    movie S3. Cross-sectional thick filament fly-through.

    movie S4. Ribbon structure.

    movie S5. Nonmyosin densities.

    References (94114)

  • Supplementary Materials

    This PDF file includes:

    • table S1. Sequence identity among representative myosins.
    • Modeling the paramyosin core
    • In-depth discussion of subfilaments versus ribbons
    • The IHM and possible relevance to stretch activation
    • fig. S1. Resolution of the reconstruction.
    • fig. S2. Comparison of the Fourier transform of the map to the x-ray pattern of whole muscles.
    • fig. S3. Poor fitting of the IHM with the blocked head contacting the backbone.
    • fig. S4. Orientation of the IHM in Lethocerus filaments compared to tarantula filaments.
    • fig. S5. Schematic illustration of the paramyosin modeling.
    • fig. S6. Model of the paramyosin core.
    • fig. S7. Illustration of particle classification scheme.
    • fig. S8. Key to movie S3.
    • Legends for movies S1 to S5
    • References (94114)

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • movie S1 (.mp4 format). Fitting of IHM structure into the 20 Å map.
    • movie S2 (.mp4 format). Myosin molecule within the filament.
    • movie S3 (.mp4 format). Cross-sectional thick filament fly-through.
    • movie S4 (.mp4 format). Ribbon structure.
    • movie S5 (.mp4 format). Nonmyosin densities.

    Files in this Data Supplement:

Navigate This Article