Three dimensional electron microscopy (3DEM) is a set of techniques for determining the structures of large proteins and protein complexes. It has been shown that cryogenic EM (cryo-EM) has the ability to determine structures to high enough resolution to unambiguously place side chains in the EM density. Though cryo-EM has the potential for atomic resolution, a number of hurdles are present in order to achieve high-throughput structure determination of low-symmetry and no symmetry complexes. Here we propose to develop software tools for high-throughput high-resolution 3DEM. In the first aim, we propose to develop software tools for single particle 3DEM including software for automated specimen screening, automated data acquisition parameter optimization, and an infrastructure for processing helical samples. In the second aim, we propose to develop tools for tomographic data collection and processing including acquisition of dual axis tilt series, and developing and infrastructure for processing tomographic subvolumes. This research will benefit human health by facilitating the determination of protein structures that are relevant for human disease.
This project proposes to develop software for 3D electron microscopy (3DEM) to facilitate high- resolution high-throughput structure determination of proteins and protein complexes that are important to human health. These tools are needed to overcome current roadblocks to protein structure determination by 3DEM. This benefits human health by improving structure determination so that researchers can better understand the molecular basis of diseases.
| Mendez, Joshua H; Stagg, Scott M (2018) Assessing the quality of single particle reconstructions by atomic model building. J Struct Biol 204:276-282 |
| Paraan, Mohammadreza; Bhattacharya, Nilakshee; Uversky, Vladimir N et al. (2018) Flexibility of the Sec13/31 cage is influenced by the Sec31 C-terminal disordered domain. J Struct Biol 204:250-260 |
| Heymann, J Bernard; Marabini, Roberto; Kazemi, Mohsen et al. (2018) The first single particle analysis Map Challenge: A summary of the assessments. J Struct Biol 204:291-300 |
| Stagg, Scott M; Mendez, Joshua H (2018) Processing apoferritin with the Appion pipeline. J Struct Biol 204:85-89 |
| Mukhitov, Nikita; Spear, John M; Stagg, Scott M et al. (2016) Interfacing Microfluidics with Negative Stain Transmission Electron Microscopy. Anal Chem 88:629-34 |
| Spear, John M; Noble, Alex J; Xie, Qing et al. (2015) The influence of frame alignment with dose compensation on the quality of single particle reconstructions. J Struct Biol 192:196-203 |
| Noble, Alex J; Stagg, Scott M (2015) Automated batch fiducial-less tilt-series alignment in Appion using Protomo. J Struct Biol 192:270-8 |
