SAXS STUDIES OF ACETOACETATE DECARBOXYLASE TOWARD CRYSTAL STRUCTURE DETERMINATIO

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 乙酰乙酸脱丙烯酰酶是第一个进行机理研究的酶，在丙酮生产中具有重要的生物技术意义。 获得的分辨率为9.8的EM图显示了这种十二聚体酶的极不寻常的组装-具有D 6对称性的中空球体，直径为100埃，由所有β折叠组成。 我们记录溶液X射线散射数据有两个主要目的：1）记录准确的散射强度，用于校正EM数据中的对比度传递函数; 2）获得基于溶液的结构包络。EM或SAXS包络线将被用作初始相位模型，以使用我们的1.8 ½分辨率单晶衍射数据获得晶体结构。我们从我们的解X射线散射数据中获得了回转半径43，最大尺寸117（。这些值与其十二聚体结构（29 kDa单体）完全一致。cryoEM结构已相应地缩放，以获得晶体结构测定中的初始相。通过运行Gasbor，从Q~1.0 <$-1的复合低角和高角X射线散射数据推导出的溶液结构包络的初步解释是一个圆柱形结构，具有圆形边缘，中间有一个中心空腔，其中有一个6重对称轴贯穿。我们正在改进我们的结构渲染方法，以获得更高分辨率的结构。

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.