Structural proteomics towards studying protein complexes in cellular context

Recent advances in cryo-electron microscopy (cryo-EM) allow for rapid and direct visualization of high-resolution three-dimensional (3D) structures. Unfortunately, current methods are often limited to identification of and structure determination for highly purified protein samples, preventing characterization of protein complex structures in their native form. Being able to determine a protein structure in the native biological “habitat” by imaging, for example, an unpurified cell lysate, is essential to understanding its role in the mechanisms that drive biological processes. Methods such as shotgun-electron microscopy (shotgun-EM) and cryo-electron tomography (cryo-ET) aim to study structural biology in the biological context of the cell, which allows for the definition of native interaction partners and identification of spatial information. However, a major and frequent tradeoff of imaging biological assemblies in a near-native context is a decrease in structure resolution. Integrating cryo-EM with complementary techniques such as mass spectrometry can allow for characterization of protein architectures without purifying for a specific target. Furthermore, protein-protein interactions identified by mass spectrometry or other computational methods, in conjunction with cutting-edge methods for 3D structure determination and prediction, can actually be used to investigate the architecture of multiple distinct protein complexes from a single mixture. Here, I detail computational methods that I have developed for determining protein interaction interfaces based on surface complementarity. This serves as a useful tool for determining how protein subunits may fit together within an EM map. I then describe an approach for using intermolecular evolutionary couplings between amino acids interfaces to assemble proteins into intermediate- to low-resolution EM maps. Because both of these methods are dependent on reliable protein structure, I then used in situ cross-linking mass spectrometry (XL/MS) to evaluate and improve the quality of AlphaFold predicted protein structures. Finally, I present the use of these methods to study native ciliary protein architectures, such as the intraflagellar transport A (IFT-A) complex. Ciliopathies are a class of diseases that arise from dysfunction in cilia. I show that using integrative methods to study the IFT-A architecture allows for the molecular interpretation of disease-associated alleles. The development and integration of these methods provides a toolbox for studying ciliary assemblies in their native environment and interpreting these lower resolution assemblies with the goal of providing a blueprint for the molecular basis of disease.Cellular and Molecular Biolog