This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Bacteriophage EL, a member of the Myoviridae family of bacteriophages, is a massive phage whose genome is 211,215 base pairs in length and has 201 predicted open reading frames. This phage encodes its own chaperonin and is the first chaperonin ever found to be encoded by a virus. The function of a chaperonins in general is to assist in the proper folding of denatured or misfolded proteins. Chaperonins are also ATPases in that they hydrolyze ATP and use the resulting energy to aid in the protein folding. Chaperonins can have one of three different conformational states. The open form, with both ends open, a half-open form with one end open and a closed form with both ends closed off. Each of the conformational states is determined by the binding of ATP or ADP to the nucleotide binding pocket. There are also large conformational changes that occur upon binding of the substrate, which demonstrate that conformational changes occur not only upon binding and hydrolysis of ATP but upon binding of the substrate protein to be folded. Our lab has determined the structure of the EL virus chaperonin open conformation to 7 angstrom resolution by cryo-electron microscopy. The closed and half open conformations are currently at about 11 angstrom resolution each and are still improving with additional refinement cycles. We are now attempting to solve the structure to atomic resolution using x-ray crystallography. Preliminary data collection on our home source has produced diffraction to 4 angstrom resolution. We anticipate reaching better than 3 angstrom resolution using synchrotron radiation. Molecular replacement solutions have proven unsuccessful because know structures, namely bacterial GroEL, have an amino acid identity of less than 20%. Furthermore, the cryo-EM maps of the virus encoded chaperonin have shown tremendous differences in structure compared to bacterial GroEL. Collecting native and heavy atom data will therefore help us solve the structure to atomic resolution.
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