# Altering electron-induced radiolysis to optimize cryo-EM/ET imaging

> **NIH NIH R01** · UT SOUTHWESTERN MEDICAL CENTER · 2024 · $344,400

## Abstract

PROJECT SUMMARY
Living cells have a complex and often precise organization in space and time. Determining the three-
dimensional structure of proteins and other biomolecules, as well as understanding how they form functional
networks in vivo, is a major goal of modern biology. Answering these questions is paramount to understanding
both the normal functions of proteins, as well as their dysfunctions. Cryo-electron microscopy (cryo-EM) and
cryo-electron tomography (cryo-ET) are powerful imaging tools that enable visualization and structural
determination of native macromolecular complexes in vitro and in situ. Researchers use cryo-EM to resolve
isolated (macro)molecules at near-atomic or atomic resolution, whereas cryo-electron tomography can
visualize macromolecules and organelles inside unperturbed cells with molecular to near-atomic resolution.
Together, cryo-EM and cryo-ET have the potential to reveal a more comprehensive and detailed (atomic-level)
picture of the spatiotemporal organization and inner workings of cells. However, to fully realize the potential of
cryo-EM/ET imaging techniques, we need new tools and approaches that can address outstanding technical
limitations, such as radiation damage of frozen-hydrated biological specimens and the localization of specific
molecules in cryo-tomograms. Therefore, we will develop a new sample preparation strategy that can reduce
electron-induced radiolysis of frozen-hydrated specimens, thereby improving the resolution of cryo-EM/ET
images and/or the speed of structure determination (Aim 1). Additionally, we plan to develop a cloneable,
hyper-bubbling protein tag that would allow the precise localization of target proteins in otherwise noisy, and
difficult-to-parse, cryo-tomograms (Aim 2). We will then apply these new tools to biological model systems (i.e.
rapidly frozen and cryo-FIB milled E. coli and yeast cells) as proof of principle. The successful fulfillment of our
research aims will further enhance the revelatory power of cryo-EM/ET techniques and illuminate the complex
and dynamic relationship between molecular structure and function.

## Key facts

- **NIH application ID:** 10869343
- **Project number:** 1R01GM154131-01
- **Recipient organization:** UT SOUTHWESTERN MEDICAL CENTER
- **Principal Investigator:** Daniela Nicastro
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $344,400
- **Award type:** 1
- **Project period:** 2024-09-04 → 2026-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10869343

## Citation

> US National Institutes of Health, RePORTER application 10869343, Altering electron-induced radiolysis to optimize cryo-EM/ET imaging (1R01GM154131-01). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10869343. Licensed CC0.

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