Molecular mechanism of the bacterial flagellar axial coupling joint

1.鞭毛轴向蛋白及其复合物的x射线晶体学研究(1)HAP3的26 kDa片段的晶体结构以1.9 Å分辨率（PDB ID:2D4X）进行了解析。(2)以2.1 Å分辨率（PDB ID:2D4Y）解析HAP1 49 kDa片段的晶体结构。(3)我们找到了一个稳定的HAP2-HAP3-FlgN复合物，并建立了该复合物的纯化流程。(4)我们获得了HAP1-FlgN复合物的小晶体。从钩到细丝的鞭毛轴向结构分析。(1)通过将31 kDa FlgE片段的x射线结构拟合到电子冷冻显微镜得到的密度图中，构建了hook的原子模型。该模型和随后的仿真研究揭示了分子万向节的机理。(2)钩丝结的结构基于HAP1和HAP3的晶体结构、丝和钩模型，建立了钩丝结的局部结构模型。该结构清楚地揭示了如何将机械上截然不同的两种结构耦合并平稳传递扭矩的分子机理。(3)轴向结构的分子动力学模拟对鞭毛细丝模型进行了两种不同类型的分子动力学模拟研究。通过大规模MD仿真，确定了控制能量受挫和多态转变的开关相互作用。从粗粒度的MD模拟中证明了溶剂对多晶转变的影响。(1)利用纯化的FlgG蛋白和短钩片段重建了鱼钩的远端鱼竿。(2)我们从一个多棒突变株中纯化了基体，并用电子冷冻显微镜在8.1 Å分辨率下分析了多棒的结构。

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.