SAXS STUDIES OF ACETOACETATE DECARBOXYLASE TOWARD CRYSTAL STRUCTURE DETERMINATIO

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 乙酰乙酸酯脱羧酶是第一个从机理上进行研究的酶，对丙酮的生产具有重要的生物技术意义。获得的9.8？分辨率的EM图显示了这种十二聚体酶的一个非常不寻常的组装-一个具有D6对称性的中空球，直径100埃，由所有的β-折叠组成。我们记录了溶液X射线散射数据，主要用于两个目的：1)记录准确的散射强度，用于修正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包络将被用作初始相模型，利用我们的1.8°分辨率的单晶衍射数据来获得晶体结构。我们从我们的溶液X射线散射数据中得到了回转半径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。为了在晶体结构测定中获得初始相，相应地对CryoEM结构进行了定标。通过运行Gasbor对Q~1.0？-1的复合低角和高角X射线散射数据，初步解释了溶液结构包络是一个圆边圆柱形结构，中间有一个中心空腔，其中有一个6重对称轴贯穿其中。我们正在完善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.