Tomographic 3D Reconstruction of Quick-Frozen, Ca2+-Activated Contracting Insect Flight Muscle

Abstract

Motor actions of myosin were directly visualized by electron tomography of insect flight muscle quick-frozen during contraction. In 3D images, active cross-bridges are usually single myosin heads, bound preferentially to actin target zones sited midway between troponins. Active attached bridges (approximately 30% of all heads) depart markedly in axial and azimuthal angles from Rayment's rigor acto-S1 model, one-third requiring motor domain (MD) tilting on actin, and two-thirds keeping rigor contact with actin while the light chain domain (LCD) tilts axially from approximately 105 degrees to approximately 70 degrees. The results suggest the MD tilts and slews on actin from weak to strong binding, followed by swinging of the LCD through an approximately 35 degrees axial angle, giving an approximately 13 nm interaction distance and an approximately 4-6 nm working stroke.

Mechanistic role of movement and strain sensitivity in muscle contraction

“…Our kinetic scheme resembles mechanisms in which myosin heads are enabled by movement to generate tension after an L-jump, a class of model first proposed by Huxley and Kress in the mid 1980s (44). We find a compelling match between our kinetics and a structural mechanism based on 3D images of individual attached crossbridges obtained from electron micrographs of flash-frozen contracting insect flight muscle (33). Here, lever arm domains of heads attached to thin filaments occupy a wide sweep of angles from an antirigor angle of 125°to a rigor-like end-of-stroke angle of 70°, an angular motion equivalent to an Ϸ13-nm swing of the lever arm, a sequence illustrated in the diagrams of Fig.…”

“…A step release activates mobile low-stiffness AMD I cross-bridges by movement (red arrow) while simultaneously discharging tension-generating AMD II /AMD III isometric cross-bridges (blue arrow). Diagrams based on 3D tomograms of contracting insect flight muscle (32,33) show both axial and azimuthal catalytic and lever arm domain movement during step 6 activation. After activation, the lever arm domain alone appears to change angle during the power stroke (steps 7 and 8).…”

A cell-centered database for electron tomographic data

“…The breadth of these reconstructions is approaching that of light microscopy, while still revealing the exquisite, high-resolution detail typically found in the best electron micrographs of ultrathin sections. Electron tomography can also be used to resolve the substructure of individual macromolecules and so provides a natural bridge between molecular and cellular imaging methods (Harlow et al, 1998(Harlow et al, , 2001McEwen and Frank, 2001;Taylor et al, 1997Taylor et al, , 1999. Researchers are employing tomography not only to investigate the structure of purified protein complexes, but also to investigate their structure in situ, fitting the lower resolution tomographic data with atomic models or molecular envelopes obtained by molecular microscopy and X-ray crystallography (McEwen and Frank, 2001;Taylor et al, 1999).…”

“…Electron tomography can also be used to resolve the substructure of individual macromolecules and so provides a natural bridge between molecular and cellular imaging methods (Harlow et al, 1998(Harlow et al, , 2001McEwen and Frank, 2001;Taylor et al, 1997Taylor et al, , 1999. Researchers are employing tomography not only to investigate the structure of purified protein complexes, but also to investigate their structure in situ, fitting the lower resolution tomographic data with atomic models or molecular envelopes obtained by molecular microscopy and X-ray crystallography (McEwen and Frank, 2001;Taylor et al, 1999). Others are using the superior resolution of cell-level tomograms to search for molecular signatures of proteins in complex cellular environments based on an understanding of protein structure, without the need for protein-specific stains (Bohm et al, 2000;Koster et al, 1997).…”

Electron Tomography: A 3D View of the Subcellular World

“…Frozen specimens often are freeze-substituted, resin-embedded, and contrasted with heavy metals, which deposit around cellular components. Although this process may limit the resolution and prevent detection of secondary-structure elements within the proteins, it is sufficient to provide the shapes of large molecular complexes (9).…”