Electron Microscopic Studies of Helical Polymers

II A new real-space method of electron microscopic reconstruction is used to study the structures of several helical protein polymers including filamentous bacteriophage fd, bacterial EspA filaments, archaeal Methanococcus maripaludis pili, bacterial F-pili, RecA-DNA-RecB nuc filaments and von Willbrand Factor (VWF) tubules. We report the first image reconstruction of a filamentous virus, bacteriophage fd, by electron cryomicroscopy. The result shows that new computational approaches to helical reconstruction can greatly extend the ability to visualize heterogeneous protein polymers at a reasonably high resolution. The EspA filaments in enteropathogenic E. coli belong to the Type III Secretion System. We find that these filaments have a switching between the more compressed and extended filaments in the packing of putative alpha-helices around the hollow lumen. The flexible structure allows these filaments to maintain their function in a mechanically harsh environment. We show that archaeal Methanococcus maripaludis pili have two different subunit packing arrangements: C 4 symmetry and one-start helical symmetry. Remarkably, both schemes appear to coexist within the same filaments. This result has many implications for understanding the evolutionary divergence of bacteria and archaea. Exchange of DNA between bacteria involve conjugative pili. We solve the longexisting ambiguity in the packing geometry of F-pilin subunits. C 4 symmetry and onestart helical symmetry were identified within the same filament. The E. coli RecBCD enzyme facilitates the loading of RecA onto the singlestranded DNA. Three-dimensional reconstructions suggest that RecB nuc binds to the III ATP-binding core of RecA (conserved in RadA and Rad51), with a displacement of RecA's C-terminal domain. Since the RecA C-terminal domain has been shown to be regulatory, we suggest that the RecA interaction observed is part of the loading mechanism where RecB displaces the RecA C-terminal domain and activates a RecA monomer for polymerization. The VWF tubules bind connective tissue and mediate platelet adhesion at sites of vascular injury. The reconstruction shows a 1-start helix with 4.2 subunits per turn with a variable axial rise. The symmetry and location of interdomain contacts suggest that decreasing pH along the secretory pathway coordinates the disulfide-linked assembly of VWF multimers with their tubular packaging. Dedication IV To my Grandfather Jiyou, because you led me into the world of sciences. To Dad and Mom, who unconditionally love me in every minute of my life. Finally, to my husband Weiping, without whom I could not have done this.

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