Molecular mechanism of the bacterial flagellar axial coupling joint

1.鞭毛轴蛋白及其配合物的X射线结晶学研究(1)HAP3的26 kDa片段在1.9Å分辨率下的晶体结构(PDB ID：2D4X).(2)HAP1的49 kDa片段的晶体结构在2.1Å分辨率下(PDBID：2D4Y).(3)我们发现了一个稳定的HAP2-HAP3-FlgN配合物，并建立了该配合物的纯化程序.(4)得到了HAP1-FlgN的小晶体.钩的结构分析：(1)钩的结构。通过将31 kDa的FlgE片段的X射线结构与电子冷冻显微镜获得的密度图进行拟合，建立了钩的原子模型。该模型和后续的模拟研究揭示了分子万能连接的机理。(2)钩丝结的结构我们根据HAP1和HAP3的晶体结构、灯丝和钩模型建立了钩丝结的局部结构模型。轴向结构的分子动力学模拟对鞭毛丝模型进行了两种不同类型的分子动力学模拟研究。从大规模的分子动力学模拟中确定了控制能量受挫和多态转变的开关相互作用。3.杆和杆-钩复合体的结构分析(1)我们用纯化的FlgG蛋白和短钩片段从钩上重建了远端杆。(2)我们从一株多杆突变株中提纯了基体，并用电子冷冻显微镜分析了分辨率为8.1Å的多杆的结构。

1.X-ray crystallographic studies of the flagellar axial proteins and their complexes(1) Crystal structure of a 26 kDa fragment of HAP3 was solved at 1.9 Å resolution (PDB ID:2D4X).(2) Crystal structure of a 49 kDa fragment of HAP1 was solved at 2.1 Å resolution (PDB ID:2D4Y).(3) We found a stable complex of HAP2-HAP3-FlgN and established a purification procedure of the complex.(4) We obtained small crystals of the HAP1-FlgN complex.2. Analyses of the flagellar axial structure from the hook to the filament.(1) Structure of the hookAn atomic model of the hook was constructed by fitting the X-ray structure of a 31 kDa fragment of FlgE into the density map obtained from electron cryo-microscopy. The model and following simulation studies revealed the molecular universal joint mechanism.(2) Structure of the hook-filament junctionWe built the partial structural model of the hook-filament junction, based on the crystal structures of HAP1 and HAP3, the filament and the hook model. The structure clearly indicated the molecular mechanism of how to couple the mechanically distinct two structures and to transmit torque smoothly.(3) Molecular dynamics simulation of the axial structureTwo different types of molecular dynamics simulation studies were conducted on the flagellar filament model. Switch interactions which control the energy frustration and polymorphic transition were identified from a massive MD simulation. Solvent effects on the'polymorphic transition were demonstrated from the coarse-grained MD simulations.3.Structure analysis of the rod and the rod-hook complex(1) We have reconstructed the distal rod from the hook using purified FlgG proteins and the short hook fragments.(2) We purified the basal body from a poly rod mutant strain and analyzed the structure of the poly rod at 8.1 Å resolution by electron cryo-microscopy.