Three-dimensional structure of frozen-hydrated paracrystals of myosin rod

Abstract

Myosin rod, the tail fragment of myosin, aggregates into various structures at physiological ionic strength. One form, the type II paracrystal, a thin, ribbon-like species, has recently been studied using conventional electron microscopy and was shown to possess two-dimensional order and, further, is likely to be useful in the investigation of the arrangement of myosin molecules in the backbone of the vertebrate muscle thick filament (Ward & Bennett, 1989). We have now examined this aggregate in the frozen-hydrated state by cryo-electron microscopy. Image analysis indicated that the projected structure has the same p12, plane group symmetry as seen after negative staining. A three-dimensional map, calculated from projections, indicated that the structure comprises a bilayer arrangement of strands of grouped rod molecules, with the strands parallel in each layer. The layers themselves are related by screw symmetry. Strands in adjacent layers have opposing polarity with their long axes at an angle of 32 degree to each other. Protein density measurements, carried out on unstained specimens using electron energy-loss spectroscopy, showed that the 44 X 13 X 13 nm unit cell is composed of 40% protein. The density measurements indicated that 9-12 rod molecules pass through each strand. Modelling rod molecules with 43 nm parallel overlaps in a body-centered tetragonal lattice produced a strand that compared favorably with the reconstructed strand. The size and content of these strands suggests that they are analogous to subfilaments observed in the vertebrate myosin thick filament.

Mutations at the same amino acid in myosin that cause either skeletal or cardiac myopathy have distinct molecular phenotypes

“…6) [15–21]. As expected, WT LMM formed paracrystals with 14.0 nm periodicity, similar to the 14.3 nm axial spacing seen for muscle fibers in vivo [15, 16, 19, 21]. Neither R1500P nor R1500W alters the periodicity of the paracrystals, with each being similar to WT and indicating that the gross morphology of filaments formed from these mutants is unaffected (Table 3).…”

Mutations in the β-myosin rod cause myosin storage myopathy via multiple mechanisms

“…Paracrystals are well-ordered protein assemblies that provide an established model for thick filament formation (1,(20)(21)(22)(23)(24)(25)(26). Similarly to light scattering experiments, proteins were dialyzed from a high salt buffer into a buffer with physiological salt concentration allowing them to assemble into well-ordered arrays.…”