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Abstract:
A great deal of information on the 3-dimensional structure of the protein assemblies involved in muscle contraction has been obtained using conventional transmission electron microscopy. In recent years, developments in cryo-electron microscopy have facilitated work with fully hydrated, non-chemically fixed specimens. It is shown how this technique can be used to visualize muscle sarcomere filaments in quasi-native conditions, to access hitherto inaccessible states of the crossbridge cycle, and to obtain new high resolution structural information on their 3-dimensional protein structure. A short introduction to the crossbridge cycle and its biochemically accessible states illustrates the problems amenable to studies using the electron microscope, as well as the possibilities offered by cryo-microscopy on vitrified samples. Work on vitrified cryo-sections and myosin filament suspensions demonstrates the accessibility of crossbridge states and gives implications on the gross structural features of myosin filaments. Recent studies on actin filaments and myosin (S1) decorated actin filaments provide the first high resolution data on vitrified samples. The use of photolabile nucleotide precursors allows the trapping of short lived states in the millisecond time range, thereby visualizing intermediate states of the crossbridge cycle.
