Woldeyes, R. A. | Search bioRxiv

bioRxiv2023

Cryo-electron tomography reveals the structural diversity of cardiac proteins in their cellular context

Cardiovascular diseases are a leading cause of death worldwide, but our understanding of the underlying mechanisms is limited, in part because of the complexity of the cellular machinery that controls the heart muscle contraction cycle. Cryogenic electron tomography (cryo-ET) provides a way to visualize diverse cellular machinery while preserving contextual information like subcellular localization and transient complex formation, but this approach has not been widely applied to the study of heart muscle cells (cardiomyocytes). Here, we deploy an optimized cryo-ET platform that enables cellular-structural biology in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Using this platform, we reconstructed sub-nanometer resolution structures of the human cardiac muscle thin filament, a central component of the contractile machinery. Reconstructing the troponin complex, a regulatory component of the thin filament, from within cells, we identified previously unobserved conformations that highlight the structural flexibility of this regulatory complex. We next measured the impact of chemical and genetic perturbations associated with cardiovascular disease on the structure of troponin. In both cases, we found changes in troponin structure that are consistent with known disease phenotypes--highlighting the value of our approach for dissecting complex disease mechanisms in the cellular context.

biophysics↗

bioRxiv2019

Comparing serial X-ray crystallography and microcrystal electron diffraction (MicroED) as methods for routine structure determination from small macromolecular crystals

Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal electron diffraction (MicroED) have emerged as useful methods for obtaining structural information from crystals on the nanometer to micron scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges not encountered in traditional macromolecular crystallography experiments. Here, we describe XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A (CypA). Our results provide a roadmap for researchers hoping to design macromolecular microcrystallography experiments, and they highlight the strengths and weaknesses of the two methods. Specifically, we focus on how the different physical conditions imposed by the sample preparation and delivery methods required for each type of experiment effect the crystal structure of the enzyme.

biophysics↗