Revealing Molecular Adversaries of Human Health Using Advanced Imaging Technology

Single particle electron microscopy (EM) allows us to examine the molecular world and gain insights into protein structures implicated in human disease. Visualizing the 3D architecture of the macromolecules can inform drug design and preventative care. While X-ray crystallography and NMR are able to resolve atomic structures, the methodology is better suited for smaller structures with limited flexibility. Single particle EM allows us analyze larger structures that have inherent flexibility. Protein structures can broadly be categorized as symmetry or asymmetric. There are computational advantages when analyzing symmetrical structures. Specifically, structural information can be extrapolated from fewer vantage points. Thus, symmetrical macromolecules are an advantageous for pioneering new methodologies in single particle EM. Rotavirus double layered particles (DLPs) are large macromolecular complexes that display icosahedral symmetry. Previous studies have led to a high resolution structure of transcriptionally inactive rotavirus frozen in time. However, to more fully understand rotavirus we need to examine the structure under transcriptionally active conditions. To expand our understanding, we first evaluated these viral assemblies using cryo-EM under active and inactive conditions. We found both internal and external structural differences. Based on these findings we sought to further our understanding of these nano-machines by developing a liquid cell environment to evaluate their dynamics over time. Our research not only developed a new methodology to evaluate active particles over time, we also found that the mobility of the DLPs were directly correlated to the level of transcriptional activity. When analyzing asymmetrical and flexible protein complexes previous studies have utilized methodologies to limit the proteins’ conformational variability. While this does allow for a higher resolution structure, it limits our understanding to a specific orientation and compromises the biological insights. BRCA1 is an asymmetric protein containing a large flexible region and is important in the prevention of breast cancer. We utilize silicon nitride microchips with integrated wells and decorated with a lipid monolayer to capture and image BRCA1 complexes. This imaging platform minimizes heterogeneity and ensures the sample quality while not biasing confirmation. Thus, allowing for high resolution cryo-EM imaging of flexible native proteins. We were able to examine BRCA1 complexes from cells at both the primary and metastatic sites. Our ability to visualize these proteins in their native form provide insights into the variability of BRCA1 in disease progression. We found that BRCA1 complexes isolated from metastatic cells have additional density in the C-terminal domain. Our data suggests this density it due an interaction with p53. Overall, our methodologies highlight the power of single particle EM for studying protein complexes. Furthermore, our findings emphasize the importance of examining protein complexes in their native state. Revealing Molecular Adversaries of Human Health Using Advanced Imaging Technology A. Cameron Varano GENERAL AUDIENCE ABSTRACT Single particle electron microscopy (EM) allows us to examine the molecular world and gain insights into protein structures implicated in human disease. Visualizing the 3D architecture of macromolecules can inform drug design and preventative care. While X-ray crystallography and NMR are able to resolve atomic structures, the methodology is better suited for smaller structures with limited flexibility. Single particle EM allow us analyze larger structures that have inherent flexibility. Protein structures can broadly be categorized as symmetry or asymmetric. There are computational advantages when analyzing symmetrical structures. Specifically, structural information can be extrapolated from fewer vantage points. Thus, symmetrical macromolecules are an advantageous for pioneering new methodologies in single particle EM. Rotavirus double layered particles (DLPs) are large, highly symmetrical macromolecular complexes that represent an ideal model system for developing technology. Previous studies have led to a high resolution structure of inactive rotavirus DLP frozen in time. However, to more fully understand rotavirus we need to examine the structure under active conditions. To expand our understanding, we first evaluated these viral assemblies using cryo-EM under active and inactive conditions. We found structural differences. Based on these findings we sought to further our understanding of these nano-machines by developing a liquid cell environment to evaluate their dynamics over time. Our new methodology revealed new insights into the mobility of the DLPs. When analyzing asymmetrical and flexible protein complexes previous studies have utilized methodologies to limit the proteins’ movement. While this does allow for a higher resolution structure, it limits our understanding to a specific orientation and compromises the biological insights. BRCA1 is a highly flexible asymmetric protein implicated in the development of breast cancer. We utilize specialized microchips to capture and image BRCA1 complexes. This imaging platform ensures sample quality and allows for high resolution cryoEM imaging of flexible native proteins. We were able to examine BRCA1 complexes from cells at both the primary and metastatic sites. Our ability to visualize these proteins in their native form provide insights into the variability of BRCA1 in disease progression. Our data found that BRCA1 complexes isolated from metastatic cells are structurally different than those at the primary site. Overall, our methodologies highlight the power of single particle EM for studying protein complexes. Furthermore, our findings emphasize the importance of examining protein complexes in their native state.