Introduction & Background

For a biochemist the usual way to encounter macromolecular complexes of a cell is by handling small amounts of solutions, photometric values or spots on a polyacrylamid gel. In contrast to that it was a truly extraordinary experience for me to directly ‘see’ the actual activity of ion channels by patch-clamping, a method I learned before starting as a postdoc in Günter Blobel’s lab in New York. There, however, literally seeing such complexes by electron microscopy (EM) became even more fascinating. As a result, instead of performing electrophysiological experiments I started to study the protein-conducting channel, the Sec61 complex, by EM. In all cells this universally conserved membrane protein complex mediates the membrane translocation or insertion of most secretory and membrane proteins. Reliable sorting of such proteins to their final destination inside or outside the cell is a vital step of gene expression affecting up to 30 % of all genes. These proteins carry a so-called signal sequence at their N-terminus that is recognized by the signal recognition particle (SRP). Upon binding of the signal sequence SRP targets the ribosome, the protein synthesis machinery, via the membrane-bound SRP receptor (SR) to the Sec61 complex. Here, the ribosome continues translation while translocation or membrane insertion takes place. Since this process involves very large and dynamic macromolecular machines we use cryo-electron microscopy (cryo-EM) in combination with single particle reconstruction to investigate the structural basis of protein sorting, translocation, membrane insertion and folding.