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Three-Dimensional Reconstruction of Thick Filaments from Rapidly Frozen, Freeze-Substituted Tarantula Muscle

https://doi.org/10.1006/jsbi.1995.1049Get rights and content

Abstract

We have applied three-dimensional helical reconstruction techniques to images of myosin filaments of tarantula leg muscle obtained from rapidly frozen, freeze-substituted specimens. Computed Fourier transforms of filaments selected from longitudinal sections show up to six layer lines indexing on the 43.5-nm helical repeat of myosin crossbridges. The three-dimensional reconstruction, performed after separation of overlapped Bessel functions, shows four continuous strands of density on the surface of the filament, modulated by density at 14.5-nm intervals, corresponding to the myosin heads aligned approximately along the helical strands. In transverse view, the reconstruction shows four projections and is similar in profile to myosin filaments seen in thin transverse sections of rapidly frozen muscle. The reconstruction is similar to that of negatively stained, isolated tarantula filaments except that in the latter there is an additional modulation of the helix density, which better resolves the two heads of each myosin crossbridge. Thus, the general arrangement of the myosin heads in the freeze-substituted specimens is preserved, although finer details of structure such as individual myosin heads are lost.

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Cited by (12)

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    These results were in general agreement with theoretical models proposed earlier (Squire, 1973). Helical Fourier-based three-dimensional (3D) reconstructions of myosin thick filaments have been calculated for several invertebrates like Limulus (Stewart et al., 1981, 1985), tarantula (Crowther et al., 1985; Padrón et al., 1995), scorpion (Stewart et al., 1985), scallop (Vibert and Craig, 1983) and vertebrates like frog (Stewart and Kensler, 1986). However, these reconstructions of negatively stained thick filaments did not allow unambiguous resolution of the two myosin heads and the myosin subfragment 2 (S2) due to their relatively low (5–7 nm) resolution.

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    Following freeze-substitution, specimens are embedded and sectioned conventionally. Using this approach, it has been possible to demonstrate the helical organization of myosin heads in thick filaments in situ in tarantula muscle and to determine directly the rotational symmetry of the head organization in both tarantula and scallop muscle by observing thick filaments in transverse section [73–75]. Similar detail has also been observed in vertebrate muscle [76–78] leading to improved insights into vertebrate sarcomeric structure.

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    They are thus appropriate for biochemical, enzymatic, and structural studies. Tarantula muscle was used in this study because of our interest in the molecular organization of the myosin heads in this muscle in the relaxed state and in the structural and biochemical changes that occur in the myosin heads when they are phosphorylated (Crowther et al., 1985; Craig et al., 1987; Padrón et al., 1992, 1995; Offer et al., 2000; Hidalgo et al., 2001). These studies have been hampered by the numerous actin filaments in cryo-electron microscopy (cryo-EM) images of crude filament suspensions (much higher than in specimens examined by negative staining), which interfere greatly with thick filament image analysis.

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