In Detail

Three-dimensional reconstructions provide accurate geometric and spatial representations of macromolecular complexes found at synapses.

Determining the 3-D structure of CaM kinase II (CaMKII) is one of our key endeavors. We accomplish this through 3-D reconstruction of single particle images collected by stain and cryo-electron microscopy. These efforts are in collaboration with Dr. Pawel Penczek.

We have successfully accomplished this goal, and a low resolution structure (23 A) is shown in Figure 1 (see Kolodziej et al., 2000). This recombinant form of the neuronal (alpha) isoform of CaMKII is a twelve-subunit complex. The catalytic domains reside in foot-like processes that extend away from a central gear-shaped core formed by the C-terminal domains of each subunits. We have also solved the structure for the other three (beta, gamma and delta) CaMKII isoforms which all show similar structures to the alpha isoform (see Gaertner et al., 2004).                                                                                                                                                                                               

It is possible to significantly increase the resolution of these 3-D reconstructions. Our group recently obtained a Polara 300 kV FEG electron microscope equipped with a 4 K x 4 K Teitz CCD camera. The improved quality of data from this instrument along with significantly larger sets of data could lead to reconstructions of less than 10 A.

We are interested in understanding the structural basis for the autophosphorylation mechanism (present data indicates this occurs through nearest neighbor interactions) and whether significant architectural rearrangement might occur when the kinase is activated.

Post-Synaptic Densities Analyzed with Immuno-gold Labeling and Cryo-EM tomography

Post-synaptic densities (PSDs) are a specialized organelle found at the apical region of spines at synapses in the mammalian brain.  They are responsible for organizing the receptors and signaling molecules in the post-synaptic compartment in apposition to the neurotransmitter release machinery on the pre-synaptic side of the synapse.  While the molecular composition of PSDs have been studied extensively using biochemical, immunochemical and proteomic approaches, how the individual molecules are spatially organized in the PSD is largely unknown.  We have undertaken a systematic quantitative analysis of the distribution of individual molecules within the PSD by using immunochemical approaches and electron microscopy.