# Developing minimal purification cryo-EM to understand mitochondrial myopathies

> **NIH NIH R21** · SCRIPPS RESEARCH INSTITUTE, THE · 2023 · $444,109

## Abstract

ABSTRACT
Single particle cryoEM structure determination is now a widely used methodology that has revealed the detailed
mechanisms underlying a wide range of biological systems. High-resolution single particle cryoEM studies have
helped us understand how environmental or genetic factors perturb normal biological function, and how these
factors can give rise to disease. Insights gained through such structural studies of cellular machinery have greatly
benefited drug discovery efforts, as well as expanded our understanding of drug resistance and therapeutic
relapse. However, successful single particle cryoEM structure determination continues to be dependent on the
production and purification of highly homogeneous, biochemically stable samples for imaging. Here, we plan to
harness the unique strengths of single particle cryoEM technologies - minimal sample requirements and an
exceptional capacity for structural characterization of highly heterogeneous data - to move beyond this traditional
approach. Precedence for such studies have been set by previous high-resolution cryoEM structures that were
determined from heterogeneous mixtures of soluble or membrane-associated proteins extracted from single-cell
lysates. We plan to extend these approaches to elucidate structures of endogenous mammalian mitochondrial
complexes. In particular, the methodologies developed by this work will establish an avenue to perform structural
investigation of mitochondrial complexes derived from mitochondrial myopathy patients. Mitochondrial
dysfunction in skeletal muscle cells can have severe pathological outcomes, and is associated with a variety of
muscle-wasting diseases and numerous neuromuscular disorders. One in 5000 individuals in the U.S. suffers
from mitochondrial myopathies due to genetic mutation, and while substantial effort has been placed on
understanding the genetics of these diseases, we lack an underlying molecular description of the specific
perturbations responsible for pathology. Directly visualizing the endogenous mitochondrial complexes that carry
mutations implicated in disease states enables us to inspect how missense mutations impact macromolecular
assembly and interactions. We will develop mitochondrial isolation and structure determination methodologies
to enable detailed structural assessment of the endogenous complexes involved in human mitochondrial
proteostasis and the mitochondrial OXPHOS system, without the need for extensive protein purification. We
have shown that mitochondrial lysates can be directly applied to EM grids and imaged to yield high-resolution
structures of abundant complexes. We will further develop this pipeline to produce high-resolution structures of
mitochondrial complexes and interaction partners from the distinct mitochondrial subcompartments, providing
important molecular insights into how mutations associated with mitochondrial myopathies perturb protein
structure and function. The results will advance our understand...

## Key facts

- **NIH application ID:** 10732697
- **Project number:** 1R21AR083157-01
- **Recipient organization:** SCRIPPS RESEARCH INSTITUTE, THE
- **Principal Investigator:** Gabriel C Lander
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $444,109
- **Award type:** 1
- **Project period:** 2023-09-01 → 2025-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10732697

## Citation

> US National Institutes of Health, RePORTER application 10732697, Developing minimal purification cryo-EM to understand mitochondrial myopathies (1R21AR083157-01). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10732697. Licensed CC0.

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