Single particle electron microscopy in combination with mass spectrometry to investigate novel complexes of membrane proteins

Large data sets of molecular projections of the membrane proteins Photosystem I and Photosystem II from cyanobacteria were analyzed by single particle electron microscopy (EM). Analysis resulted in the averaging of 2D projections from the purified complexes but also in the simultaneous detection and averaging of 2D projections from large contaminating complexes, which were present in frequencies as low as 0.1%. Among them T-shaped and L-shaped contaminants were found. The L-shaped particles could be assigned to Complex I just from the shape, although no Complex I from a cyanobacterium has been structurally characterized. A systematic comparison by single particle EM and mass spectrometry of two differently purified Photosystem II complexes resulted in the assignment of PsbZ, a small peripheral subunit of 6.8 kDa, within the structure. Together these data suggest that screening for membrane protein structures by single particle EM and mass spectrometry may be a new approach to find novel structures of such proteins. We propose here a scheme for searching for novel membrane protein structures in specific types of membranes. In this approach single particle EM and mass spectrometry, after pre-fractionation using one- or multidimensional protein separation techniques, are applied to characterize all its larger components.