UNDERSTANDING MUSCLE CONTRACTION BY COMBINING CRYSTALLOGRAPHY, CRYO-EM AND FIBRE DIFFRACTION. K.C. Holmes, Max Planck Institute for Medical Research, Postfach 103820, D-69028 Heidelberg, Germany

Muscle contraction comes about via the relative sliding of two sets of protein filaments, the "thick" - myosin-containing and "thin" - actin-containing filaments. The relative movement is brought about by the "Cross-bridges" which project out from the thick filaments and which by means of an asynchronous cyclical "rowing action" and concomitant hydrolysis of ATP shift the actin passed the myosin.

The atomic structure of the actin filament has been determined by combining the protein crystallographic structure of the actin monomer (1) with fiber diffraction patterns from orientated gels of actin filaments (2). The problem of refinement of this structure is discussed by Tirion [Topic 03.04]. The Structure of the myosin cross-bridge has been determined by protein crystallography and the structure of the acto-myosin complex has been determined by combining the structures of the actin filament and the myosin cross bridge with the help of cryo-electron microscopic reconstructions from actin filaments carrying a myosin cross-bridge atttached to each actin (so-called "decorated actin") (3, 4). The myosin cross bridge is "tadpole-like" in shape. the head (sometimes called the motor domain) binds to the actin whereas the tail (sometimes called the regulatory domain) does the rowing. There is a cleft in the motor domain which is thought to provide the link between ATP and actin.

The myosin cross bridge is necessarily polymorphic: to understand muscle contraction one needs to understand how the shape of the cross bridge responds to the binding and hydrolysis of nucleotides [Rayment, Topic 04.13] and to the binding of actin. EM studies of the actomyosin complex show that the tail rotates on binding ADP [Milligan, Topic 04.13]. Since the complex between myosin cross-bridge and monomeric actin has not been crystallised it is imperative to get high resolution data from cryo EM reconstructions. One method of extending the resolution is by the use of an energy-filter microscope. Images with a resolution of 15-20Å have been obtained which allow a more detailed examination of the effect of actin-binding on the myosin cross-bridge. Fiber diffraction from orientated gels of decorated actin in the presence and absence of ADP can also be used to register changes in the actomyosin structure.

1. Kabsch et al (1990) Nature 347:37-44.

2. Holmes et al (1990) Nature 347:44-49.

3. Rayment et al (1993) Science 261:58-65.

4. Schröder et al (1993) Nature 364:171-174.