We will judge homogeneity and screen favorable nucleotide excision repair complexes assembly condition first by negative stain EM. A resolution of 15 Å is essentially the limit for negative stain EM and will justify an expectation of near-atomic resolution by cryo-EM. If we do reach 15 Å, we will proceed with cryoEM, taking advantage of the new, direct electron detector available on campus. We will collect 10,000-100,000 particle images in different orientations to enable image classification, orientation, and 3D reconstruction through iterative projection mapping by the projection-slice theorem. Structures will be validated, for example, by tilt-pair analysis, subunit localization by antibody labeling or fusion of large globular protein domain such as Maltose Binding Protein (MBP), and ultimately the unambiguous placement of known atomic structures. A resolution of 10 Å would start revealing duplex DNA and protein helices. If we fall short of near-atomic resolution, sub-nanometer resolution would still allow unambiguous docking of previously determined atomic structures or homology models into the EM density.
|Effective start/end date||9/13/16 → 8/31/21|
- University of California, Lawrence Berkeley National Laboratory (7336107 Mod. 4 // 5P01CA092584-18)
- National Cancer Institute (7336107 Mod. 4 // 5P01CA092584-18)