Three-dimensional asymmetric reconstruction of tailed bacteriophage.
A universal goal in studying the structures of macromolecules and macromolecular complexes by means of electron cryo-microscopy (cryo-TEM) and three-dimensional (3D) image reconstruction is the derivation of a reliable atomic or pseudoatomic model. Such a model provides the foundation for exploring in detail the mechanisms by which biomolecules function. Though a variety of highly ordered, symmetric specimens such as 2D crystals, helices, and icosahedral virus capsids have been studied by these methods at near-atomic resolution, until recently, numerous challenges have made it difficult to achieve sub-nanometer resolution with large (≥~500Å), asymmetric molecules such as the tailed bacteriophages. After briefly reviewing some of the history behind the development of asymmetric virus reconstructions, we use recent structural studies of the prolate phage ϕ29 as an example to illustrate the step-by-step procedures used to compute an asymmetric reconstruction at sub-nanometer resolution. In contrast to methods that have been employed to study other asymmetric complexes, we demonstrate how symmetries in the head and tail components of the phage can be exploited to obtain the structure of the entire phage in an expedited, stepwise process. Prospects for future enhancements to the procedures currently employed are noted in the concluding section.
Tang
Jinghua
J
Department of Chemistry & Biochemistry, University of California, San Diego, La Jolla, California, USA.
Sinkovits
Robert S
RS
Baker
Timothy S
TS
D001435:Bacteriophages / Q000648:ultrastructure*; D020285:Cryoelectron Microscopy / Q000379:methods*; D021621:Imaging, Three-Dimensional / Q000379:methods*; D046529:Microscopy, Electron, Transmission / Q000379:methods