Cryo-electron microscopy on ribosome-functional complexes

Studying stalled nascent chain complexes
A tunnel traverses the complete width of the large ribosomal subunit (~100 Å in length) and is thought to be the conduit that the nascent chain uses to exit out of the particle. Although the passage of the nascent chain through the ribosomal tunnel was first believed to be a passive process, evidence is accumulating that indicates that the nascent chain may interact with the tunnel to regulate translation as well as influence the fate of the de novo synthesised protein.
We have determined the structure of a ribosomal nascent chain complexes by cryo-EM; the latter being performed by in collaboration with the group of Dr. David Stuart and Prof. Knud Nierhaus. Our analysis by cryo-EM revealed that while many of the changes observed in the ribosome are independent of the nascent chain being translated, there were specific changes localised within the ribosomal tunnel indicating that (i) the ribosomal tunnel has undergone some very dramatic conformational changes, localised in the vicinity of the nascent chain and/or (ii) the additional material represents the nascent chain itself.

Ribosomal subunit assembly factor complexes
Another essential aspect to the regulation of translation is the pathway involving the synthesis and assembly of the ribosomal subunits themselves – pathways that are poorly understood in bacteria, let alone in the more complex eukaryotic organisms, such as humans. In collaboration with the group of Prof. Yokoyama , we have assembled stable homogenous complexes of the small GTPase Era bound to the 30S subunit from T. thermophilus, which have been analysed using cryo-EM in collaboration with the group of Dr. Agrawal. This reconstruction provides the first direct visualization of the binding site of an important factor necessary for ribosome assembly.
The presence of Era homologues in eukaryotes suggests that the mechanism by which Era participates in ribosome assembly may be generally applicable to all kingdoms of life.

Ribosome functional complexes
The MPI-MG has recently acquired a helium-cooled Tecnai G2 Polara cryo-electron microscope enabling in-house examination of ribosome functional complexes. Future projects undertaking using this machine will be done in collaboration with Prof. Christian Spahn at the Charite, Berlin .