bioRxiv2025
Low-angle X-ray diffraction is a powerful technique for analyzing the molecular structure of the myofilaments of striated muscle in situ. It has contributed greatly to our understanding of the relaxed, 430-[A]-repeating organization of myosin heads in thick filaments in skeletal and cardiac muscle. Using X-ray diffraction, changes in filament structure can be detected on the [A] length scale and millisecond time scale, leading to models that are the foundation of our understanding of the structural basis of contraction. As with all X-ray fiber diffraction studies, interpretation requires modeling, which has previously been based on low-resolution knowledge of thick filament structure and is complicated by the contributions of multiple filament components to most X-ray reflections. Here, we use an atomic model of the human cardiac thick filament C-zone, derived from cryo-EM, to compute objectively the contributions of myosin heads, tails, titin, and cMyBP-C to the diffraction pattern, by including/excluding these components in the calculations. Our results support some previous interpretations but contradict others. We confirm that the myosin heads are responsible for most of the intensity on the myosin layer-lines, including the M3 meridional. Contrary to expectation, we find that myosin tails contribute little to the pattern, including the M6 meridional; this reflection arises mainly from heads and other components. The M11 layer line (39 [A] spacing) arises mostly from the curved and kinked structure of titin, which allows eleven [~]42-[A]-long domains to fit into the 430 [A] repeat. The M11 spacing can be used as a measure of strain in the myosin filament backbone as there is negligible head contribution. These insights should aid future understanding of the X-ray pattern of intact muscle in different conditions such as contraction and drug treatment. Significance statementX-ray diffraction is widely used to study the structure of striated muscle, revealing the molecular organization of the thick and thin filaments in situ. Changes in the X-ray pattern during contraction provide insights into contractile mechanisms on the [A] length scale and millisecond timescale. Interpretation of X-ray patterns is based on modeling, which is complicated by contributions of multiple filament components to different reflections and the lack of a reliable thick filament model. Here, we use a cryo-EM-based atomic model of the thick filament to compute contributions of different filament components to the diffraction pattern, by including/excluding these components in the calculations. The insights gained will aid interpretation of the X-ray pattern in relaxation and contraction and following drug treatment.