Many important biological macromolecules exist as helical polymers. Examples are actin, tubulin, myosin, RecA, Rad51, flagellin, pili, and filamentous bacteriophage. The first application of three-dimensional reconstruction from electron microscopic images was to a helical polymer, and several laboratories today are using helical tubes of integral membrane proteins as specimens for solving the near-atomic structure of these proteins in the electron microscope. We have developed a method to analyze and reconstruct electron microscopic images of macromolecular helical polymers. We can show that when there is disorder or heterogeneity, when the specimens diffract weakly, or when Bessel functions overlap, we can do far better with our method than can be done using traditional approaches. In many cases, structures that were not even amenable to analysis can be solved at fairly high resolution (better than 10 A) using our method. Disseminating this method widely, and further developing it, will require effort along several lines. We need to create procedures to establish whether structures are unique and correct, and we can show that ambiguities that are present in structure determination at limited resolution have not been sufficiently appreciated. We will need to implement the algorithm within a more user-friendly package of EM image analysis tools, so that use of the method does not require extensive familiarity with the SPIDER system. We will optimize the method for the particular geometries and problems of lipid tubes containing integral membrane proteins. At the same time, effort will be put into exploring a real-space algorithm for the alignment of images and refinement of a structure. All of the methods development will take place using a number of systems that are of great biological and clinical interest. These include filamentous bacteriophage, widely used in laboratories as cloning vectors and for phage display, pili from pathogenic bacteria, shown to be essential for bacterial infectivity, Type III secretion system needles from Enteropathogenic E. coli, and F-pili, involved in bacterial conjugation and the spread of antibiotic resistance within a bacterial population. Most of these projects will thus have an impact on understanding structures involved in bacterial pathogenesis and disease. The methods that we will be developing and disseminating will have an even larger impact in many areas of NIH-funded research, from understanding muscle structure and dystrophies to infectious diseases. ? ? ?
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