Experimental protein structure determination methods make up a fundamental part of our understanding of biological systems. Manual interpretation of the output from these methods has been made obsolete by the sheer size and complexity of the acquired data. Instead, computational methods are becoming essential for this task and with the advent of high-throughput methods the efficiency and robustness of these methods are a major concern. This work focuses on the computational challenge of efficiently extracting statistically supported information from noisy or significantly reduced experimental data.

Small-angle X-ray scattering (SAXS) is a method capable of probing structural information with many experimental benefits compared to alternative methods. However, the acquired data is a noisy reduction of a large set of structural features into a low-dimensional signal-mixture, which significantly limits its interpretability. Due to this SAXS has this far been limited to conclusions about large-scale structural features, like radius of gyration or the oligomeric state of the sample. In this thesis I present an approach where SAXS data is used to guide molecular dynamics simulations to explore experimentally relevant conformational states. The experimental data is fed into the simulations through a metadynamics protocol, which explores the experimental data through conformational sampling subject to thermodynamic restraints. I show how this approach makes it possible to use SAXS to produce atomic-resolution models and make further-reaching conclusions about the underlying biological system, in particular by showcasing de novo folding of a small protein.

Another experimental method that generates noisy and reduced data is cryogenic electron microscopy (cryo-EM). Due to recent development in the field, the computational burden has become a considerable bottleneck, which greatly limits the throughput of the method. I present computational techniques to alleviate this burden through the use of specialized algorithms capable of efficient execution on graphics processing units (GPUs). This work improves the computational efficiency of the entire pipeline by several orders of magnitude and significantly advances the overall efficiency and applicability of the method. I show how this enables the development of improved algorithms with increased capabilities for extracting relevant biological information form the data. Several such improvements are presented that significantly increase the resolution of the refinement results and provide additional information about the dynamics of the system. Additionally, I present an application of these methods to data collected on a biogenesis intermediate of the mitochondrial ribosome. The new structures provide insights into the timing of the rRNA folding and protein incorporation as well as the role of two previously unknown assembly factors during the final stages of ribosome maturation.

Mitochondria, popularly known as powerhouse of the cell, contain specialised mitoribosomes that synthesise essential membrane proteins. These essential proteins are required to form enzyme complexes, which carry out the process of oxidative phosphorylation (OXPHOS). OXPHOS is carried out by five enzyme complexes (Complex I-V), out of which complex I, III and IV pump protons during electron transfer from NADH to O₂ and complex V uses the generated proton gradient to synthesise ATP. Cryo-EM, as a revolutionary technique in structural biology made it possible to determine the structures of mitoribosome assembly intermediates and respiratory chain supercomplexes. These structures have allowed us to investigate the mitoribosome biogenesis pathway in human and yeast and to gain deeper insights into the architecture of supercomplexes. In the first area of research, using cryo-EM we were for the first time able to capture mitoribosomes in different late stages of assembly and to determine their high-resolution structures with novel factors bound. Investigation of this process was previously unreachable due to lack of techniques to trap these mitoribosome complexes in different states of assembly. The structures of these assembly intermediates establish the role of assembly factors such as MALSU1, LOR8F8, mt-ACP, MTG1 and mitoribosomal proteins (MRPs) in mitoribosome biogenesis and to ensure proper maturation of each subunit, reflecting their role in regulating translation. Furthermore, genetic deletion studies of MTG1 and uL16m in yeast show the importance of transiently acting factors and MRPs in the mitoribosome assembly process and their effects on translation. The assembly pathway of mitoribosomes is critical for protein synthesis since defects in the translation process causes inherited human pathologies. Therefore, elucidation of mitoribosomal biogenesis pathways may also contribute to the development of potential new therapeutic opportunities. In the second research area, structures of the respiratory chain supercomplex from yeast were determined. These are the first near-atomic resolution structures that show organization of complex III and complex IV into two distinct classes that form higher order assemblies (III₂IV₁and III₂IV₂). Moreover, the architecture of the supercomplex structures differs from the previously determined respirasomes (I₁III₂IV₁) structures in mammals. We obtained a near-atomic resolution structure of the yeast complex IV, revealed core protein-protein and protein-lipid interactions that hold the supercomplex together. Moreover we found novel subunits required for supercomplex formation in S. cerevisiae. The last part of my study focuses on cryo-EM sample method development where we could successfully demonstrate the usefulness of a simple pressure-assisted sample preparation method for microcrystals, proteins and mitochondria. Our findings show great resolution improvements of selected area electron diffraction patterns of microcrystals, a significant reduction in needed sample concentration for single particle studies and an enrichment of gold nano-particles for tomographic studies.