Xie X, Rao S, Walian P, Hatch V, Phillips G N, Cohen C
Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02254.
J Mol Biol. 1994 Mar 4;236(4):1212-26. doi: 10.1016/0022-2836(94)90022-1.
Electron microscope images of highly ordered spermine-induced microcrystals of tropomyosin have been analyzed to determine the packing of the molecular filaments. Negatively stained microcrystals terminate in a distinctive "double fringe", which reveals the location of the molecular ends. This information, together with the symmetry of the structure in projection, shows that the microcrystals can be accounted for by a packing scheme of four layers of molecules in the unit cell. Knowing the position of the symmetry elements relating the layers then allows the three-dimensional space group of the microcrystals to be established as C222(1). Using cryo-electron microscopy and simulation studies, the run of the filaments and their packing in the C222(1) form have been shown to be related to those in the spermine-induced C2 crystal of tropomyosin whose structure has been solved to 9 A by X-ray crystallography. This result allows us to infer the location of the molecular ends in the C2 crystal as well, and this inference has been confirmed by analysis of thin sections of the C2 crystal. The C222(1) microcrystal has also been shown to be closely related to the classical divalent cation tropomyosin paracrystal. Based on knowledge of the molecular packing in the divalent cation paracrystal, the polarity of the molecules has been deduced in the other two crystal forms. The tropomyosin filament packing in all these forms may be accounted for by coiled-coil close packing and specific cationic bridging of negatively charged zones on the molecule. Taken together the results reveal a hierarchy of interactions in these close-packed crystalline forms, whose principles may apply to the packing in other fibrous proteins. This study also shows the usefulness of co-ordinating results from cryo-electron microscopy with negative staining in the structure analysis of such ordered arrays, and how these findings can complement the results of low resolution X-ray crystallographic studies.
