Computational Methods for In-Situ Structure Determination using Cryo-ET

Understanding how proteins interact within the cell to perform specific functions is a central goal of modern biology and crucial for understanding the diverse roles these molecules play in biomedicine. Single-particle cryo-electron tomography (SP-cryo-ET) is currently the only technology capable of visualizing macromolecules in their native environment at high resolution. Despite recent advances in sample preparation, data acquisition, and image processing, structural analysis by SP-cryo-ET is still largely confined to naturally abundant targets—such as ribosomes or ordered supramolecular assemblies—or to resolutions that are insufficient to reveal molecular-level interactions. Furthermore, the complexity and high computational demands of SP-cryo-ET workflows for data processing have restricted access to the technology for structural biologists. These challenges present a significant barrier to fully unlocking the potential of SP-cryo-ET in advancing our understanding of how proteins interact within cells to carry out specific functions and the essential roles they play in biology and disease. Building on recent progress made by our group, the goal of this project is to develop methods to address remaining bottlenecks in the SP-cryo-ET workflow that will result in broader applicability and improved access. Our long-term goal is to enable the routine visualization of a wide range of biomedically important targets in the cellular context at near-atomic resolution. By working closely with a network of experimental collaborators, we will ensure that our method development efforts are driven by diverse and impactful biological projects. Ultimately, our tools will help expand the applicability of SP-cryo-ET and accelerate its adoption, empowering structural biologists to tackle new questions regarding molecular interactions and cellular mechanisms. Project Number: 1R35GM163737-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Alberto Bartesaghi | Institution: DUKE UNIVERSITY, DURHAM, NC | Award Amount: $482,750 | Activity Code: R35 | Study Section: Special Emphasis Panel[ZRG1 MBBC-A (55)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11330742