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The thick filament is a key component of sarcomeres, the basic force-generating and load-bearing unit of striated muscle1. Mutations in thick filament proteins are associated with familial hypertrophic cardiomyopathy and other heart and muscle diseases2, 3. Despite this central importance for sarcomere force generation, it remains unclear how thick filaments are structurally organized and how its components interact with each other and with thin filaments to enable highly regulated muscle contraction. Here, we present the molecular architecture of native cardiac sarcomeres in the relaxed state, determined by electron cryo-tomography. Our reconstruction of the thick filament reveals the three-dimensional organization of myosin heads and tails, myosin-binding protein C (MyBP-C) and titin, elucidating the structural basis for their interaction during muscle contraction. The arrangement of myosin heads is variable depending on their position along the filament, suggesting that they have different capacities in terms of strain susceptibility and activation. Myosin tails exhibit a distinct arrangement and pattern of interactions. These are likely orchestrated by three alpha and three beta titin chains that are arranged like a spring, suggesting the existence of specialized roles of thick filament segments in length-dependent activation and contraction. Surprisingly, while the three titin alpha chains run along the entire length of the thick filament, titin beta does not. The structure also demonstrates that the C-terminal region of MyBP-C binds myosin tails and unexpectedly also directly interacts with the myosin heads, suggesting a previously undescribed direct role in the preservation of the myosin OFF state. Furthermore, we visualize how MyBP-C forms links between thin and thick filaments. These findings establish a robust groundwork for forthcoming research endeavors aiming to explore muscle disorders that involve sarcomeric structural components.

Disease-causing bacteria use a variety of secreted toxins to invade and subjugate their hosts. While the machinery responsible for secretion of many smaller toxins has already been established, it remains enigmatic for larger ones such as Tc toxins from human and insect pathogens, which approach the size of a prokaryotic ribosome. In the present study, we combine targeted genomic editing, proteomic profiling and cryo-electron tomography of the insect pathogenYersinia entomophagato reveal that a specialized subset of bacterial cells produces the Tc toxin YenTc as part of a complex toxin cocktail released into the environment by controlled cell lysis using a transcriptionally-coupled, pH-dependent type 10 secretion system (T10SS). Our results dissect the process of Tc toxin export by a T10SS in hitherto unprecedented detail, identifying that T10SSs operate via a previously unknown lytic mode of action, and establishing them as crucial players in the size-insensitive release of cytoplasmically folded toxins. With T10SSs directly embedded in Tc toxin operons of major human pathogens such asYersinia pestisandSalmonella enterica, we anticipate our findings to model an important aspect of pathogenesis in bacteria with a significant impact on global human health.

Nebulin is a major structural protein of skeletal sarcomeres and is essential for proper assembly and contraction of skeletal muscle1. It stabilises and regulates the length of thin filaments,2 but the structural mechanism remains nebulous. Using electron cryotomography and sub-tomogram averaging, we present the first structure of native nebulin bound to thin filaments within the A-band and I-band of intact sarcomeres. This in-situ reconstruction reveals unprecedented detail of interaction at pseudo-atomic resolution between nebulin and actin, providing the basis for understanding the structural and regulatory roles of nebulin. The position of nebulin on the thin filament indicates that there is no contact to tropomyosin or myosin, but an unexpected interaction with a troponin-T linker, possibly through two binding motifs on nebulin. In addition, our structure of myosin bound to the thin filaments reveals different conformations of the neck domain, both within the same sarcomere and when compared to purified structures, highlighting an inherent structural variability in muscle. We provide a complete description of cross-bridge formation on fully native, nebulin-containing thin filaments at near-atomic scale. Our structures establish the molecular basis for the role of nebulin as a thin filament “molecular ruler” and the impact of nemaline myopathies mutations that will aid future development of therapies.

Sarcomeres are the force-generating and load-bearing devices of muscles. A precise molecular understanding of how the entire sarcomere is built is required to understand its role in health, disease and ageing. Here, we determine thein situmolecular architecture of vertebrate skeletal sarcomeres through electron cryo-tomography of cryo-focused ion beam-milled native myofibrils. The reconstructions reveal the three-dimensional organisation and interaction of actin and myosin filaments in the A-band, I-band and Z-disc and demonstrate how α -actinin cross-links antiparallel actin filaments to form a mesh-like structure in the Z-disc at an unprecedented level of molecular detail. A prominent feature is a so-far undescribed doublet of α-actinin cross-links with ∼ 6 nm spacing. Sub-volume averaging shows the interaction between myosin, tropomyosin and actin in molecular detail at ∼ 10 Å resolution and reveals two coexisting conformations of actin-bound heads. The flexible orientation of the lever arm and the essential and regulatory light chains allow the two heads of the “double-headed” myosin not only to interact with the same actin filament but also to split between two actin filaments. Our results provide new insights into the conformational plasticity and fundamental organisation of vertebrate skeletal muscle and serve as a strong foundation for futurein situinvestigations of muscle diseases.

Cryo-electron tomography (cryo-ET) is an emerging technique to study the cellular architecture and the structure of proteins at high resolution in situ . Most biological specimens are too thick to be directly investigated and are therefore thinned by milling with a focused ion beam under cryogenic conditions (cryo-FIB). This procedure is unautomated and prone to contaminations, which makes it a tedious and time-consuming process, often leading to suboptimal results. Here, we present new hardware and software tools that overcome the current limitations. We developed a new glove box and a high vacuum cryo transfer system and installed a stage heater, a cryo-shield and a cryo-shutter in the FIB milling microscope. This reduces the ice contamination during the transfer and milling process and simplifies the handling of the sample. In addition, we introduce a new software application that automates the key milling steps. Together, these improvements allow for high-quality, high-throughput cryo-FIB milling. This paves the way for new types of experiments, which have been previously considered infeasible.