In our research we try to understand how large multiprotein complexes, such as the nuclear pore complex (NPC), are built. Because of their size and complexity such molecular machines are beyond the scope of a single method and have long been a challenge for structural biology. Atomic-resolution methods such as X-ray crystallography or NMR require a purified specimen and are not suitable for very large assemblies. Although, electron microscopy allows the observation of large complexes in their native environment in the cell, it is often very difficult to assign the electron density to individual proteins. In fluorescence microscopy the identity of the protein is known and super-resolution (SR) now allows us to visualize details below the diffraction limit. When SR is combined with particle averaging, proteins' positions can be mapped to a subnanometer precision, a scale that makes light microscopy applicable to structural studies of large complexes. SR microscopy can therefore link different types of data and eventually help generate pseudo-atomic models of multiprotein assemblies. Additionally, a big advantage of especially
Super-Resolution Microscopy Gives New Insights into Nuclear Pore Complex Organization
EMBL researchers combine Ground State Depletion Microscopy with a novel image analysis method
The Nuclear Pore Complex (NPC) is a large protein complex in the nuclear membrane, representing the gate to the eukaryotic genetic makeup. This complex consists of several hundreds of proteins which form a selective gate for compounds destined to enter or leave the nucleus.
Because of this outstanding function the structure of the NPC is of great interest. So far, structural analysis of the NPC has been limited mainly to crystallization studies or electron microscopy (EM). Several structures of single components have already been deciphered by crystallography. However, the organization of individual proteins inside the complex remained elusive. One gap has now been closed by the use of super-resolution microscopy.
Jan Ellenberg and his team of scientists at the EMBL in Heidelberg have obtained new insights into the NPC structure with the help of Ground State Depletion (GSD) microscopy.
Anna Szymborska recently published the results of this research in the Science article "Nuclear Pore Scaffold Structure Analyzed by Super-Resolution Microscopy and Particle Averaging" and comments on her achievements and the potential of Ground State Depletion microscopy for protein complex analysis in the following interview.