This thesis centers on an overarching theme of mitochondrial health and disease, focusing specifically on the roles of two mitochondrial proteins, ClpXP and FARS2. These proteins are crucial for maintaining mitochondrial quality control and homeostasis, with ClpXP involved in protein degradation and FARS2 in translation. We employ structural techniques to gain a deeper understanding of the functions of these two proteins. We first utilize cryo-electron microscopy technology (cryo-EM) to elucidate the structures of ClpXP, providing mechanistic insights into its assembly, activation, and regulatory functions within the mitochondria. We then discuss our efforts in the development of graphene grids to address the challenges we encountered with ClpXP cryo-EM sample preparation. Finally, we conclude with biochemical and X-ray crystallographic studies on FARS2 variants associated mitochondrial diseases. In chapter 1, I provide an overview of the diverse functions of mitochondria and the diseases linked to mitochondrial dysfunctions. Mitochondria, the muti-functional organelles that host over a thousand different proteins, orchestrate crucial cellular processes. To ensure proper import, folding, and turnover of proteins within the mitochondria, a system of regulatory mechanisms— collectively termed mitochondrial quality control—oversees mitochondrial proteostasis. Among these mechanisms, ATPases associated with diverse cellular activities (AAA+) proteins harness energy from ATP hydrolysis to facilitate protein folding and degradation. In this chapter, I highlight the important roles played by the AAA+ superfamily of proteins in maintaining mitochondrial health, focusing particularly on human ClpXP (hClpXP). The human ClpXP complex (hClpXP) is crucial for mitochondrial protein quality control, degrading misfolded or excess proteins using ATP hydrolysis. While bacterial ClpXP is well-characterized, the molecular determinants underlying hClpXP assembly and regulation remain poorly understood. In chapter 2, we use cryo-EM to determine the structures of hClpP in isolation and in complex with hClpX, revealing how ClpX binding promotes rearrangement of an asymmetric ClpP 10 heptamer to assemble a symmetric tetradecamer. Our hClpXP structures also highlights the stabilizing role of a previously uncharacterized hClpX sequence specific to Eukaryotes, referred to as the E-loop, and its importance in enzymatic activities. We also show that the hClpXP complex adopts a proteolytically inactive conformation until introduction of a peptide mimetic, which drives hClpP to adopt a conformation compatible with proteolysis. These findings substantially advance our understanding of the molecular mechanisms defining hClpXP activation and function. hClpXP sample preparation was a major challenge in our efforts to achieve the high-resolution structures. High-resolution cryoEM requires trapping evenly distributed macromolecules in various orientations within a thin layer of vitreous ice. A common challenge with current sample preparation method is the exposure of specimens to the hydrophobic air-water interface (AWI), which results in a preferred orientation of particles in cryoEM grids due to interactions between the hydrophobic patches on biological specimens and the AWI. hClpP exhibits a strong preferred orientation in the vitrified ice, limiting our ability to obtain a high-resolution cryo-EM structure. Chapter 3 describes our efforts to develop cryo-EM graphene grids in an attempt to overcome this preferred orientation. The use of graphene grids has emerged as a promising approach to mitigate AWI interaction; however, graphene grids are expensive and complicated to produce in-house. In this chapter, we describe a detailed protocol for efficient fabrication of graphene-coated cryo-EM grids, which will benefit the cryoEM community at large. hClpXP regulates folding and turnover of proteins involved in respiratory chain function. Many of these proteins are synthesized within the mitochondria by mitochondrial aminoacyl tRNA synthetases (mt-aaRS), a group of nuclear-encoded enzymes that facilitate transfer of each of the 20 amino acids to its cognate tRNA for protein translation. Phenylalanyl-tRNA synthetase 2 (FARS2) is one such mitochondrial aaRS, responsible for transferring phenylalanine to its cognate tRNA. In chapter 4, we present clinical and molecular characterizations of novel FARS2 variants that cause neonatal mitochondrial disease. We determined the high-resolution crystal structure of FARS2 R198L variant, revealing a local structural destabilization in the catalytic domain. We 11 further show that R198L mutant has a reduced thermal stability and impaired enzymatic activity. The data culminated from this collaborative work illuminate the underlying mechanism driving the pathogenicity of FARS2 variant in causing early-onset mitochondrial epilepsy.