Myosin Crossbridge Configurations in Equilibrium States of Vertebrate Skeletal Muscle

Abstract

The positions and orientations of the myosin heads in relaxed, active, rigor and S1-labelled fish muscle are being determined by analysis both of electron micrographs and of low-angle X-ray diffraction patterns. The X-ray analysis of resting muscle makes use of the head shape defined from the study of S1 crystals, with variable head configurational parameters being used on each of the three different 3-fold symmetric 14.3 nm-spaced ‘crowns’ of myosin heads within the 42.9 nm axial repeat of the myosin filaments. Diffraction patterns were stripped using CCP13 fibre diffraction software. Searches and optimisation were carried out using simulated annealing and local refinement procedures to give a ‘best fit’ relaxed structure with a crystallographic R-factor of about 4%. It had heads oriented all the same way up (i.e. with similar rotations around their own long axes) on the myosin filament, but with a small range of axial tilts. Head configuration in rigor fish muscle is being determined by X-ray diffraction and electron microscopy of normal rigor muscle and of skinned muscle soaked with extrinsic myosin S1. Computed 3-D reconstructions of acto-S1 using X-ray amplitudes and phases from electron microscopy are informative and help to analyse the X-ray diffraction data that extend axially to about 1 nm resolution. An ambiguity is the axial direction of the observed resting myosin head array relative to the known polarity of the actin filaments. One polarity would give little axial displacement (2–3 nm) between opposite ends of the resting and rigor heads, and in this case the heads would need to rotate around their own long axes by about 115° to make a rigor attachment. The other (preferred) filament polarity would provide considerable axial swinging (14–15 nm) between the two states. We are attempting to define the absolute polarity of the resting muscle myosin head array using electron microscopy and image processing either of cryo-sections or of replicas from shadowed, freeze-fractured, rapidly frozen fish muscle fibres.