Cryo-electron microscopy (cryoEM), in the form of single particle reconstruction (SPR) and cryo-electron tomography (cryoET) analysis are rapidly growing methods for determining the three-dimensional molecular structures of biological samples. This burgeoning interest stems from the fact that the size or complexity of the biological objects that can be analyzed by the technique are only fundamentally limited by sample thicknesses on the order of a few hundred microns. Additional benefits of the SPR and cryoET techniques include (1) substantially reduced demand for sample (ng - ?g vs. >1 mg for x-ray diffraction (XRD) or nuclear magnetic resonance (NMR) analysis), (2) improved safety in instances where the sample is derived from a pathogenic organism, and (3) the potential for accelerating the rate of structure determination since development of large-scale expression systems and scaling-up of purification methods are not required. As attractive as these considerations may be, SPR and cryoET are usually medium-resolution techniques (~ 1 nm), requiring additional refinement by molecular modeling or other strategies to enhance resolution. The modest resolution obtained is, in part, due to """"""""needle-in-the-haystack"""""""" issues that arise from the labor-intensive sampling procedures that are currently used. Pandion Laboratories seeks to accelerate the productivity of these techniques by more than an order of magnitude through the development of electron microscopy grids that will concentrate the biological sample on its surface prior to cryoEM analysis. We anticipate that this simple-to-use tool will have tremendous impact on the structural biology and drug discovery communities by improving the resolution of the structures obtained via greatly enhanced sampling rates, as well as accelerating the rate of structure elucidation and extending the range of samples that can be analyzed using these techniques.
Two specific aims are proposed to achieve this goal.
AIM 1 : Develop Synthesis Pathways for Affinity Lipids. Three different classes of poly(ethylene glycol)-modified affinity lipids (i.e., metal-affinity, glycoside and antibody fragment) will be prepared on the 100 mg scale for use as the capture chemistry component in the EM grid coatings.
AIM 2 : Develop robust deposition strategies for coating electron microscopy grids with affinity lipid films. Two different deposition techniques will be evaluated for their ability to prepare affinity grids and their performance evaluated by cryoEM using a histidine-tagged protein standard.
Structure-based drug design has emerged as a powerful tool for improving the efficacy, and lowering the development costs, of new pharmaceutical ingredients. This drug discovery approach involves an iterative process of refining both the architecture and potency of a drug candidate by enhancing drug-molecular target interactions in a lock &key fashion based on high-resolution structures that have been determined for their complexes. The proposed project seeks to develop novel tools that will greatly accelerate the cryo-electron microscopy-based structure elucidation process and the accompanying structure-based drug design efforts.
|Benjamin, Christopher J; Wright, Kyle J; Hyun, Seok-Hee et al. (2016) Nonfouling NTA-PEG-Based TEM Grid Coatings for Selective Capture of Histidine-Tagged Protein Targets from Cell Lysates. Langmuir 32:551-9|