# Tools for Enabling Challenging Structure Determination by Cryo-EM

> **NIH NIH R01** · FLORIDA STATE UNIVERSITY · 2023 · $449,295

## Abstract

Abstract
Recent technological innovations have significantly increased the efficiency of cryogenic electron
microscopy (cryo-EM), causing a proliferation in the number of structures resolved with ever-increasing
resolution. As many of the "low hanging fruit" structures have been determined, studies have shifted
toward increasingly complex specimens, pushing the capabilities of current processing software and
requiring novel methodologies. One class of challenging molecule for cryo-EM is tubular molecules that
can have ambiguous symmetries. In particular, membrane remodeling proteins that form tubules are
often only locally ordered making them intractable for structure determination. Another challenge for
cryo-EM structure determination is the problem of the air/water interface. Often when blotting a sample
for cryo-EM preparation, the sample can interact with the air/water interface causing protein
denaturation, aggregation, or preferred orientations. We have developed a new technique that we are
calling reconstruction of average subtracted tubular regions (RASTR) that has the potential to solve
both of these problems. RASTR breaks down tubular cryo-EM samples into individual surfaces which
enables structure determination without knowing or applying helical symmetry and classification of
tubular samples that are only locally ordered with no long-range order. Here we propose two aims to
make RASTR more robust, generalizable, and higher-resolution. These aims will be driven by
challenging tubular samples that have proven to be intractable to typical cryo-EM structure
determination methods. Finally, a third aim is to extend the RASTR approach to single particles bound
to tubular substrates in order to protect them from the air/water interface and prevent preferred
orientations. Together the proposed aims give RASTR the potential to become a general platform to
enable structure determination of challenging samples.

## Key facts

- **NIH application ID:** 10744600
- **Project number:** 1R01GM148734-01A1
- **Recipient organization:** FLORIDA STATE UNIVERSITY
- **Principal Investigator:** SCOTT M STAGG
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $449,295
- **Award type:** 1
- **Project period:** 2023-09-19 → 2027-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10744600

## Citation

> US National Institutes of Health, RePORTER application 10744600, Tools for Enabling Challenging Structure Determination by Cryo-EM (1R01GM148734-01A1). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10744600. Licensed CC0.

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