Native architecture of the chloroplast protein-import channel

该项目旨在直接从天然膜在分子水平上破译TOC-TIC系统的蛋白质转运机制。该项目非常及时地与天然质谱8 -9和冷冻断层扫描10 -12技术的最新发展相结合，这些技术直接从天然脂质环境中分析样品，而没有化学干扰。这些技术相对较新，并带来了重大挑战。从双方的角度来看，该项目的总体目标是开发方法，以解决细胞生物学的一个重大挑战，具有根本的重要性和广泛的应用。分子生物学技术包括克隆、PCR、DNA和RNA提取、蛋白纯化、SDS-PAGE、Western Blotting（JRB）;植物遗传学和标记转基因株系的产生（JRB、PJ）;超声波处理富集复合物（JRB）;质谱法，包括自下而上和自上而下（JRB、CVR）; cryo-ET和FIB-SEM（PZ和CM）;数据和统计分析;质谱法（评估复杂光谱并进行定量分析）（JRB、CVR）cryo-ET和FIB-SEM（亚断层图像平均、3D图像重建和定量图像分析）（PZ和CM）。

This Project aims to decipher the mechanisms of protein transport by the TOC-TIC system at a molecular level directly from native membranes. This Project is very timely with the recent developments made in native mass spectrometry8-9 and cryo-tomography10-12 techniques where samples are analysed directly from native lipid environment without chemical disruptions. These techniques are relatively new and come with significant challenges. From both Parties' perspective, the overall goal of the Project is to develop methodologies to solve one of the major challenges of cell biology that has fundamental importance and broad applications. The methods developed in this Project would be highly beneficial to other researchers working on organelle membrane complexes and help cement the relationships with the Diamond in the future.Molecular biology techniques including cloning, PCR, DNA and RNA extraction, protein purification, SDS-PAGE, Western Blotting (JRB); Plant genetics and generation of tagged transgenic lines (JRB, PJ); enriching complexes by sonication (JRB); mass spectrometry including bottom-up and top-down (JRB, CVR); cryo-ET and FIB-SEM (PZ and CM); Data and statistical analysis; mass spectrometry (evaluating complex spectra and performing quantitative analysis) (JRB, CVR) cryo-ET and FIB-SEM (subtomogram averaging, 3D image reconstructions and quantitative image analysis) (PZ and CM).