Elucidation of protein structure
A homogenously immobilised analyte will have to adopt crystal symmetry in order to contribute to diffraction, a requirement for the application of this technology for structure determination. EM and X-ray studies of viral receptors and antibodies in complex with viruses provide evidence that ordered repeating structures are able to impart order onto bound molecules as a consequence of their non-covalent interactions. Specifically, image reconstruction of decorated viruses has allowed the visualisation of virus:receptor complexes at high resolution. Crysalin will exploit the unique stability of crysalin lattices to build upon this behaviour. Specifically, the robustness of the crysalin lattice will allow work in conditions that permit extremely high concentrations of cryoprotectant reagents. This in turn will allow us to use slow annealing protocols to lower the crysalin+analyste system temperature to cryogenic temperatures. This will allow the analyte to search for and discover a lowest energy configuration that we anticipate will have crystallographic symmetry.
To date we have had success in attaching analytes to crysalin, allowing observation of bound proteins and determination of protein structure:
EM images showing protein bound to a 2D crysalin lattice with ~20Ä resolution
- First images yield GFP projection at ~20Äresolution.
- Imaging of even small targets at 8~12Ä should be possible for genetically fused targets using cryo EM.