Native architecture of the chloroplast protein-import channel

本项目旨在从分子水平上破译TOC-TIC系统直接从天然膜转运蛋白质的机制。该项目非常及时地采用了天然质谱学8-9和冷冻断层摄影术10-12技术的最新进展，这些技术直接从天然脂类环境中分析样品，而不需要化学干扰。这些技术相对较新，面临着巨大的挑战。从双方的角度来看，该项目的总体目标是开发方法来解决具有根本重要性和广泛应用的细胞生物学的主要挑战之一。本项目开发的方法将对其他研究细胞器膜复合体的研究人员非常有益，并有助于巩固未来与钻石的关系。分子生物学技术，包括克隆、PCR、DNA和RNA提取、蛋白质纯化、SDS-PAGE、Western blotting(JRB)；植物遗传学和标记转基因品系的产生(JRB，PJ)；超声波浓缩复合体(JRB)；质谱学，包括自下而上和自上而下(JRB，CVR)；冷冻-ET和FIB-SEM(PZ和CM)；数据和统计分析；质谱学(评估复杂光谱和进行定量分析)(JRB、CVR)CRYO-ET和FIB-SEM(图像下平均、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).