Native architecture of the chloroplast protein-import channel

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).