SAXS STUDIES OF ACETOACETATE DECARBOXYLASE TOWARD CRYSTAL STRUCTURE DETERMINATIO

点击翻译按钮获取中文摘要

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. Acetoacetate deacrboxylase is the first enzyme to be studied mechanistically, and bares an important biotechnology implication for producing acetone. An EM map obtained to 9.8 ¿ resolution shows a highly unusual assembly for this dodecameric enzyme - a hollow sphere with D6 symmetry, 100 angstroms in diameter made up of all beta sheet. We have recorded solution x-ray scattering data for two primary purposes: 1) to record accurate scattering intensities for the correction of the contrast transfer function in the EM data; 2) to obtain solution-based structural envelope. Either an EM or SAXS envelope would be used as an initial phase model to obtain a crystal structure, using our 1.8 ¿ resolution single crystal diffraction data. We obtained the radius of gyration 43 ¿, the maximum dimension 117 (¿3) ¿ from our solution x-ray scattering data. These values are fully consistent with its dodecameric structure (29 kDa monomer). The cryoEM structure has been scaled accordingly to obtain initial phases in the crystal structure determination. A preliminary account of the solution structure envelope, deduced from the composite low- and high-angle x-ray scattering data to Q~1.0 ¿-1 by running Gasbor, is a cylindrical structure with rounded edges with a central cavity in the middle, where a 6-fold symmetry axis runs through. We are in the process of refining our structural rendering approaches toward obtaining a higher resolution structure.