3-D Tomography of Crossbridges

In order to understand how myosin produces force, it is necessary to visualize the structure of myosin during a power stroke, as it goes through the cycle of splitting ATP and binding to actin. The atomic structures of the myosin head [subfragment 1 (S1)] without nucleotide (rigor) and of its motor domain with several ATP analogues, together with in vitro motility assays and cyro-EM using S1 and actin filaments, represent great advances toward understanding the mechanism of force production by myosin. However, none of these techniques views myosin when it is attached to actin and tethered in thick filaments and actively producing force. Our collaborative approach combines X-ray diffraction, muscle mechanics, electron microscopy (EM), 3-D image analysis and atomic modeling of myosin crossbridges in situ in fibers of insect flight muscle, the most highly ordered muscle known.

Structural changes in myosin crossbridges during active force generation are visualized in 3-D by tomography of thin sections from freeze-substituted muscle fibers slam-frozen during contraction. Glycerinated insect flight muscle of the giant waterbug, Lethocerus, is stretch-activated at pCa 5.5, but at pCa ~4.0 gives isometric high static tension (HST). Tension was recorded up to the millisecond of freezing and +/-70° tilt series were collected from 25 nm longitudinal sections. The data were combined using all Fourier terms without any symmetry or translational ordering to produce an unaveraged 3-D image that preserves the native structural variation in the specimen. This is important because myosin crossbridges in actively contracting muscle are at different points in the cycle of splitting ATP and show a wide variation in crossbridge form. The non-averaging tomograms successfully display the wide range of freeze-trapped HST crossbridge forms and angles, which contrast with the more uniform "classic ~45°" angle of bridges at the end of the power stroke in rigor, a static state of maximal crossbridge attachment that occurs in the absence of ATP. Active bridges usually contain one myosin head and bind preferentially to actin target zones midway between troponins. Two to four crossbridges bind to most target zones, indicating ~30% of total myosin heads are attached to actin. This is consistent with X-ray diffraction intensities of HST fiber bundles indicating 1.4-2.9 myosin heads bound per target zone, or ~ 30% of myosin attached to actin in the native state.

Rebuilding the crystallographic atomic structure of rigor myosin subfragment 1 (S1) to fit HST crossbridges requires bending of the light chain domain (LCD) azimuthally and axially to obtain a good fit. Target-bound bridges show a range of LCD tilt angles from "anti-rigor" (105°) through 90° to rigor (~45°). In anti-rigor angled bridges, the actin-binding motor domain (MD) must also be repositioned on actin. Modeling a full power stroke by fanning all 26 rebuilt myosin heads from one actin site shows the C-terminal K843 tracing a slanted path (~14 nm axial range coupled to ~30° azimuthal sweep). Sorted by axial height above the end of the power stroke represented by rigor S1, 60-70% of the rebuilt myosin heads cluster between 0-6 nm displacement from rigor "45° angle". Motor domains of these bridges are positioned on actin like rigor, and are probably strongly bound to actin. Thirty percent of rebuilt myosin heads cluster between 6-14 nm displacement. The motor domains of these "prepower stroke" bridges are at non-rigor positions on actin, suggesting an early weak-binding attachment to actin. The model building suggests that the working stroke of myosin crossbridges encompasses two stages: axial and azimuthal movements of the motor domain on actin, followed by axial tilting of the LCD lever arm.

From the force of the fiber and the filament number/fiber, the average force per myosin head is estimated to be ~3.6 pN if all 30% of attached heads are exerting force, or 6 pN if only 60% of 30% attached produce the force measured in the fiber. Our results on myosin crossbridges in situ suggest that force production begins as the motor domain rolls on actin from weak to strong-binding, producing a bend between the motor and LCD. A power stroke is completed by tilting of the LCD lever-arm to a ~45° rigor angle.

Contributed by Mary Reedy, Duke University Medical Center

Acknowledgements The work described is a collaborative effort led by Michael K. Reedy, with the laboratories of Ken Taylor, formerly at Duke, now at FSU in Tallahassee, for 3-D reconstructions and atomic modeling, Yale Goldman and Clara-Franzini Armstrong at U. Penn for fast freezing and fiber mechanics and Richard Tregear, MRC for X-ray analysis. The composite figure was contributed by Ken Taylor.

Reference
Taylor, KA, Schmitz, H, Reedy, MC, Goldman, YE, Franzini-Armstrong, C, Sasaki, H, Tregear, RT, Poole, K, Lucaveche, C, Edwards, RJ, Chen, LF, Winkler, H & Reedy, MK 1999 Cell, 99, 421-431.

Learn about mechanochemical steps of the cross bridge cycle

Return to the Myosin Function Page
Return to the Myosin Home Page

If you use material from the reviews at The Myosin Home Page, we ask that you cite either the home page and authors, or the appropriate source publication in your work.
Copyright 1999-2004 All rights reserved

Created Sunday, November 21, 1999