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

CaMKII Single Particle Analyses:

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 cyro-electron microscopy. These efforts are in collaboration with Dr. Pawel Penczek.

The CaMKII holoenzyme has resisted crystallization and is too large for analysis by NMR. Therefore, cryo-electron microscopy followed by 3-D reconstructions is presently the only means of producing data on the 3-D structure of CaMKII holoenzymes. We have successfully accomplished this goal, and a low resolution structure (23A) is shown in Figure 1. 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 subunit.

                                                                                                                                                                                                            Figure 1.  Click to enlarge

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. This is one of our current priorities.

Other efforts include calculating 3-D structures for:

•  the Ca2+/calmodulin-bound form of CaMKII

•  the fully active and autophosphorylated form of CaMKII

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.

We are also calculating structures for CaMKII in association with fragments of synaptic proteins that bind to the complex. These studies will advance our understanding of geometric relationships between synaptic proteins and CaMKII. Since CaMKII binds to NMDA receptors and is activated by Ca2+ influx, the spatial organization of these molecules is of significant interest. Specifically, one of our priorities is determining a structure of CaMKII in association with a fragment of the NR2B subunit of the NMDA receptor. Other association molecules under investigation include densin-180, and a -actinin. We postulate that CaMKII is a central organizing element that assembles synaptic signaling complexes, and these structural analyses will provide a high-resolution view of this organization.

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

Post-synaptic densitites (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.