Structural Cell Biology of DNA Repair Machines

Project: Research project

Project Details

Description

We will judge homogeneity and screen favorable NHEJ 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. In order to validate the structural models derived from cryo-EM studies, the very same reconstituted complexes will be used for crosslinking mass spectrometry studies.
StatusActive
Effective start/end date9/21/218/31/26

Funding

  • University of California, Lawrence Berkeley National Laboratory (NO. 7615718//2P01CA092584-21)
  • National Cancer Institute (NO. 7615718//2P01CA092584-21)

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