Advancing Cryo-EM to Study Conformational Dynamics of Macromolecular Complexes from In Vitro to In Situ

National Institute Of Environmental Health Sciences

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Abstract

In FY2025, this project advanced structural studies of macromolecular complexes across a continuum that spans purified assemblies to their native cellular environments. Using cryogenic electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET), we refined workflows that capture conformational dynamics central to processes in infection, immunity, signaling, and transport. Building on prior advances in high-throughput cryo-ET and cryo-FIBSEM for lamella preparation, we further optimized strategies for imaging assemblies that operate in complex cellular contexts. Dynamic systems investigated this year included viral glycoproteins, transporters, toxins, and ion channels. Pseudotyped models of HIV-1 Env and the SARS-CoV-2 Spike were deployed to study epitope presentation, maturation, and receptor engagement, providing rigorous test cases for new methods. Additional structures revealed the mechanism of cGAMP export by the transporter ABCC1 (Immunity, 2025), antiviral drug recognition by CNT3 (Nat Chem Biol., 2024), and the molecular basis of engineered Shiga toxin immunogens with therapeutic potential (Protein Sci., 2025). We also resolved trehalose-6-phosphate synthase from Cryptococcus neoformans (PNAS, 2024), an antifungal target, and the ion channel TRPM3, regulated by neurosteroids and anticonvulsants (Nat Struct Mol Biol., 2025). Together, these studies highlight conformational dynamics as a common principle linking diverse biological processes. Progress in method development remained central. SmartScope, our automated framework for specimen evaluation, was expanded for distributed use across microscopes. VitriFlex, our open-source robotic system for flexible sample preparation (bioRxiv, 2025; doi: 10.1101/2025.07.24.666634), enabled programmable workflows and sub-second spray-to-plunge delays for time-resolved experiments. In parallel, a collaboration with Duke University advanced complementary instrumentation for sub-second cryo-EM. We also developed a cloud-based cryo-EM platform, integrating SmartScope with GPU-driven environments (Scipion, nextPYP, cryoSPARC), providing secure and scalable resources for data collection and processing across NIH. By integrating methodological innovation with the study of macromolecular dynamics across scales, this project expands the reach of cryo-EM from purified samples to in situ cellular contexts. The results deliver mechanistic insights into infection, immunity, and cellular regulation, while producing open-source tools that strengthen structural biology capabilities at NIH, contribute to the strength of our scientific community, and ultimately improve the health of the American people.