The molecular basis for sarcomere organization in vertebrate skeletal muscle

Figure S1. Cryo-FIB-ET of isolated mouse myofibrils reveals sarcomere ultrastructure and hexagonal arrangement of filaments in the cross-section view, related to Figure 1 (A) Cartoon showing the arrangement of the SEM and FIB ion source with respect to the grid. Myofibrils are depicted in red. (B) Left: SEM image taken 45° to the plane of a grid on which myofibrils were vitrified, with red arrow heads pointing toward the myofibrils. The orange arrow head indicates the milling direction. Scale bar, 50 μm. Right: Top (taken with electron beam) and side (taken with ion beam) views of the lamella taken after milling in the FIB-SEM. The top view was taken 60° angle to the plane of the grid. The side view was taken 8° to the plane of the grid, which was also the milling angle. Scale bar, 5 μm. (C) Projection image of a lamella after FIB-milling recorded at a TEM at stage 0°. Purple arrow heads indicate M-bands of sarcomeres, while green arrow heads indicate Z-discs. Scale bar, 3 μm. (D and G) Schematic diagrams of the triangular arrangement of thick filaments in the A-band (D) and the hexagonal arrangement of thin filaments in the M-band (G). Thin and thick filaments are represented by green and…

Figure 1. Isolated mouse skeletal myofibrils imaged using electron cryotomography (A) Projection image of mouse skeletal muscle, with Z-disc, I-, M-, and A-bands visible (green, light blue, dark blue, and purple, respectively). A schematic diagram is shown below, highlighting the lateral organization and cross-links in different zones. Mitochondria (Mito) and sarcoplasmic reticulum (SR) can be identified between sarcomeres. Scale bar, 1 μm. (B) Slice through an electron cryo-tomogram spanning A- to M-band. Its representative position on a sarcomere is marked as the black box in (A) (not same sarcomere). In this region, myosin heads bound to the thin filament, myosin tails emanating from the thick filament (dark blue insets) and obscure protein densities at the M-band (purple inset) can be discerned. Scale bar, 100 nm. (C) Slice through an electron cryo-tomogram spanning I-band and Z-disc. Its representative position on a sarcomere is marked as the white box in (A) (not same sarcomere). The tail-feather-like arrangement of α-actinin molecules cross-linking thin filaments in a zig-zag manner is visible (green inset). Thin filaments in the I-band have regularly-spaced nodes corresponding to…

Figure S2. Sub-volume averaging of thick filaments, related to Figures 3 and 5 (A) Slice through a tomogram depicting three adjacent thick filaments. Scale bar, 50 nm. (B) Averaged structure from a global refinement. The map suffers from missing wedge artifacts, indicating alignment focused on the missing wedge instead of actual structural features. (C) The distribution of myosin heads when retracted to the thick filaments in the relaxed state has a C3 symmetry. We therefore performed a refinement and reconstruction applying C3 symmetry. However, this did not improve the quality of the map, indicating that the core of vertebrate thick filaments is not C3 symmetric. (D) A local refinement with restricted possible rotation angles reduced alignment errors and thereby missing wedge artifacts. This reconstruction was used as a model for thick filament in Figures 3 and 5. (E) The estimated resolution of the reconstruction in (D) is 30.4 Å using the 0.143 criterion.

Figure S3. Strategies of sub-volume averaging of the in situ actomyosin complex and different “kink” conformations in the lever arm, related to Figure 2 and STAR Methods (A) Workflow of sub-volume averaging leading to the final structures of the in situ actomyosin complex and double-head myosin. Details of the colored steps are shown in (B-G). (B) Examples of selected and discarded 2D class averages. After cleaning by 2D sorting, 21,130 out of 32,421 sub-volumes were selected for subsequent processing. (C) Initial reference and the average after the first 3D refinement with only a spherical mask with a diameter of 340 Å applied. (D) Classes of the actomyosin complex after 3D classification. Prominent myosin double head pairs in each class are indicated by red arrow heads. These pairs were later aligned to each other during merging the classes by modifying the alignment parameters. The classes in the black box were selected and used for subsequent processing. (E) Classes of the myosin double-heads after the 3D classification applied on the sub-volumes re-centered on myosin heads. The two classes in the black boxes showing clear densities for the double-head were selected for the final…

Figure 2. Sub-volume averaging of thin filaments reveals the interaction of a double-head myosin with the thin filament and two conformations of light chain domains within a double-head (A) Surface view of the in situ actomyosin structure, showing actin filament (green), tropomyosin (blue), and myosin double heads, with motor domains (yellow), essential light chains (orange), and regulatory light chains (red). Scale bar, 5 nm. (B) Close-up view of the lower myosin head and homology models based on PDB: 3I5G, 5JLH, and 6KN8. The upper 50 kDa (U50), lower 50 kDa (L50), SH3, and essential light chains (ELC) can be allocated in the map, along with part of the regulatory domain (RLC). Arrow heads indicate the interaction interfaces between actin and myosin at loop 4, helix-loop-helix motif, loop 3 of myosin (top to bottom). Arrow heads in the inset depict interaction interfaces at the cardiomyopathy loop and loop 2 (left to right). (C) Close-up of an actin subunit and structural model fitted into the EM map showing the four subdomains of an actin subunit (SD1–SD4). (D) Surface view of the structure of a complete myosin double-head including RLCs determined from averaging shifted sub-volumes…

Figure 3. Organization of the A-band in natively isolated myofibrils shows that myosin heads can adopt two interactions with thin filaments (A) XY-slices through a tomogram at three different Z positions (illustrated by the cartoon below), highlighting the myosin head densities (orange, yellow, and magenta) and the thin filament (green) apparent within the volume. Annotation of densities produces a volume in which filtered actomyosin can be fitted, shown on the right and in (B). The three myosin colors represent the contribution of the myosin head from a respective neighboring thick filament. Scale bars, 20 nm. (B) Fitted model of all thin filaments and myosin heads in a tomogram, with the black inset depicting the filament shown in (A) In the XZ view, reconstructions of corresponding thick filaments are also shown. (C) A typical cross-bridge with a myosin double-head. (D) A rare myosin split-head with two heads from the same myosin molecule binding to two different actin filaments. See also Figures S2 and S4 and Video S1.

Figure S4. Annotation and fitting of split and double heads of myosin between thin and thick filaments from A-band tomograms, related to Figures 3 and 5 (A) Annotated density of a thin filament including the myosin heads (green), with the annotated density for a myosin double-head highlighted in orange. (B) Oblique and zoomed-in view of the double-head density and its associated thick filament segmentation (pink). (C) Same view as in (B) including the fitted thin filament and myosin double-head models from sub-volume averaging. (D) Annotated density of two thin filaments including the myosin heads (green and light blue), highlighting annotated density corresponding split-head in orange. (E) Oblique and zoomed-in view of the split-head density and its associated thick filament segmentation. (F) Same view as in (E) including the fitted thin filament and myosin split-head models. The split-head is composed of two heads in the upper head conformation in order to be fitted into the density.

Figure 4. Myosin binding preference is dependent on actin orientation and distance between thin and thick filaments (A) Angular distribution of myosin heads shown by a circular histogram of the orientation of the actin subunits bound by myosin heads. The colors indicate three different thick filaments where the myosin heads originate. (B) Hotspots of myosin binding from three thick filaments shown on actin filaments. Actin filaments are colored with the footprint of the myosin heads according to multiple sequence alignment of the myosin binding profile sequences determined from the annotated volume and the fitted model in Figure 3B. See also Figure S5. The preferred side on a thin filament for binding of a certain myosin group is shown in the schematic diagram below. (C) Histograms of distances between thin and thick filaments at actin subunits with (orange) or without (green) a myosin head bound. Mean distances are 25.9 nm (SD 2.7 nm) and 26.3 nm (SD 2.7 nm), respectively. No skewness was measured in either population, meaning both distributions can be considered Gaussian.

Figure 5. Arrangement of myosin heads around a thick filament (A) Model of a thick filament with surrounding thin filament-bound myosin heads obtained from fitting into the annotated tomogram. The six different colors represent myosin heads bound to the six adjacent thin filaments. (B) Close-up view of the black inset in (A) showing varying spacings between the tips of adjacent myosin heads. (C) Different conformations of S2 fragments taken from clearly discernible annotations. (D–G) Top view of four segments of the thick filament in (A) together with the thin filaments it binds, highlighting heterogeneity of myosin head organization. Each image depicts a segment with a depth of ~40 nm. See also Figures S2 and S4 and Video S1.

Figure S5. Overall myosin binding profile generated from multiple sequence alignment of myosin binding profiles of 30 thin filaments, related to Figure 4 and STAR Methods (A) The fitted myosin head model in Figure 3B was converted to myosin binding profile sequences on thin filaments, with “R,” “G,” “B” representing actin subunits bound by three groups of myosin heads originating from three neighboring thick filaments and “E” representing myosin-free actin subunits. These sequences were aligned using multiple sequence alignment with a customized weight matrix. Only the sequence of one actin strand for each thin filament was used for alignment. The sum of occurrence of each myosin group at each actin subunit position is shown at the bottom and colored accordingly. Darker color indicates higher occurrence. (B) The colored overall myosin binding profile on both actin strands for each myosin group. Sequences of strand 2 were combined using the alignment of strand 1 shown in (A). The overall myosin binding profiles depict hotspots for myosin binding on a thin filament and the region highlighted in the black box is used to color the models shown in Figure 4B.

Figure 6. Structure and organization of thin filaments in the I-band (A) Sub-volume average of the thin filament in the I-band fitted with a homology model of the complete thin filament including troponin. Homology model is based on PDB: 6KN8. (B) 3D reconstruction of the thin filament in the I-band excluding troponin. The actin filament is depicted in green and tropomyosin in blue. At this resolution, the four subdomains of actin form a clear “U” shape (marked by the arrow head) and indicates actin barbed end faces toward the Z-disc. (C) Tropomyosin along thin filaments takes the C-state in the I-band (blue) and the M-state in the A-band (yellow). (D) XZ views of sarcomeres at the A-band and I-band highlighting the disappearance of the hexagonal pattern of thin filaments. Two hexagonal units of thin filaments and their corresponding positions in the I-band are indicated by green and orange triangles. Displacement of the filaments from A-band to I-band are shown as arrows. Asterisk denotes a filament that moved out of the field of view during the A–I transition. (E) Slice through a tomogram depicting the Z-disc and two I-bands from two adjacent sarcomeres. (F) 3D model of thin filaments…

Figure S6. Sub-volume averaging of the thin filaments in the I-band, related to Figure 6 (A and B) Comparison of tropomyosin positions in different regions of the sarcomere. Side (A) and top (B) views of a thin filament with tropomyosin in the A-band (yellow) and I-band (blue). Tropomyosin is in the M state in the A-band and resides in the C-state in the I-band. The two states differ by an azimuthal rotation of ~21°. The B-state tropomyosin from a model of Ca2+-free thin filament (PDB: 6KN7) (orange) is also shown for comparison. (C) The estimated resolution of the reconstruction of a thin filament excluding troponin in the I-band is 10.6 Å using the 0.143 criterion. (D) Slice from a tomogram showing both A-band and I-band. The solid red line represents the border between the A-band and I-band. 37-nm sections separated by the red dotted lines were selected for separate sub-volume averaging. (E) 3D view of particles of the thin filaments in the same region as (D), colored based on the state of their tropomyosin position. All A-band particles exhibit the M-state tropomyosin position (yellow). Most I-band particles exhibit the C-state tropomyosin (blue). The particles in the closest 37-nm…