# Development of Cryo-EM/TEM Technologies for Small Protein and RNA Systems

> **NIH NIH R01** · PENNSYLVANIA STATE UNIVERSITY, THE · 2020 · $546,917

## Abstract

Abstract
While a recent “resolution revolution” has catapulted the use of cryo-EM imaging for high-resolution structure
solution of biomolecules, these advances have mostly been restricted to studies of relatively large complexes
(e.g. ribosome, spliceosome, and viral particles). Properties of these complexes, such as having a molecular
weight >200 kD and inherent stability, make them ideal targets for cryo-EM, which utilizes the averaging of
data from tens/hundreds of thousands of particles to reconstruct their 3D structure. However, the vast majority
of molecules-of-interest do not have these properties, and have thus remained ignored and considered
intractable for cryo-EM studies.
Here, design and initial development of four technologies that can overcome these hurdles to make the cryo-
EM imaging practical to the research and biomedical communities are described. First, inspiration has been
taken from viral particles to produce a rigid and versatile scaffold for the capture and presentation of proteins
via a “plug-and-play” approach. Because this approach does not require genetic fusion of each new protein-of-
interest to the scaffold, “average-sized” proteins and their variants can be rapidly screened for structural study.
Here these technologies will be advanced using molecules from the PIs' field of expertise (protein/RNA
biology), but these scaffolds and methods can be applied to any functional class of proteins-of-interest.
Second, a rigid and versatile scaffold for the capture and presentation of nucleic acids has been produced.
Technologies based upon this scaffold will open the doors to cryo-EM imaging of folded RNAs that have been
heretofore considered too small for cryo-EM and transmission EM (TEM) studies. Moreover, structural studies
and measurements of the in vivo variance of RNA folding populations in cells will be determined using these
technologies.
In this proposed work, technologies will be developed for wide scale use by the community by determining and
optimizing the parameters that affect their use in cryo-EM and TEM studies of proteins and RNA. Ultimately,
these rigid, highly symmetrical, and versatile scaffolds, and the methodologies for their optimal use, will drive
down the lower practical size limits of cryo-EM and enable researchers to address a wide range of biological
questions specific to their respective fields.

## Key facts

- **NIH application ID:** 9847975
- **Project number:** 5R01GM125907-03
- **Recipient organization:** PENNSYLVANIA STATE UNIVERSITY, THE
- **Principal Investigator:** Susan Hafenstein
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $546,917
- **Award type:** 5
- **Project period:** 2018-01-01 → 2021-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9847975

## Citation

> US National Institutes of Health, RePORTER application 9847975, Development of Cryo-EM/TEM Technologies for Small Protein and RNA Systems (5R01GM125907-03). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/9847975. Licensed CC0.

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