No abstract

Studying biomolecules at atomic resolution in their native environment is the ultimate aim of structural biology. We investigated the bacterial type IV secretion system core complex (T4SScc) by cellular dynamic nuclear polarization-based solid-state nuclear magnetic resonance spectroscopy to validate a structural model previously generated by combining in vitro and in silico data. Our results indicate that T4SScc is well folded in the cellular setting, revealing protein regions that had been elusive when studied in vitro.

Forkhead box O (FOXO; DAF-16 in worms) transcription factors, which are of vital importance in cell-cycle control, stress resistance, tumor suppression, and organismal lifespan, are largely regulated through nucleo-cytoplasmic shuttling. Insulin signaling keeps FOXO/DAF-16 cytoplasmic, and hence transcriptionally inactive. Conversely, as in loss of insulin signaling, reactive oxygen species (ROS) can activate FOXO/DAF-16 through nuclear accumulation. How ROS regulate the nuclear translocation of FOXO/DAF-16 is largely unknown. Cysteine oxidation can stabilize protein-protein interactions through the formation of disulfide-bridges when cells encounter ROS. Using a proteome-wide screen that identifies ROS-induced mixed disulfide-dependent complexes, we discovered several interaction partners of FOXO4, one of which is the nuclear import receptor transportin-1. We show that disulfide formation with transportin-1 is required for nuclear localization and the activation of FOXO4/DAF-16 induced by ROS, but not by the loss of insulin signaling. This molecular mechanism for nuclear shuttling is conserved in C. elegans and directly connects redox signaling to the longevity protein FOXO/DAF-16.

1 H-detection can greatly improve spectral sensitivity in biological solid-state NMR (ssNMR), thus allowing the study of larger and more complex proteins.H owever,t he general requirement to perdeuterate proteins critically curtails the potential of 1 H-detection by the loss of aliphatic side-chain protons,which are important probes for protein structure and function. Introduced herein is al abelling scheme for 1 Hdetected ssNMR, and it gives high quality spectra for both sidechain and backbone protons,a nd allows quantitative assignments and aids in probing interresidual contacts.Excellent 1 H resolution in membrane proteins is obtained, the topology and dynamics of an ion channel were studied. This labelling scheme will open new avenues for the study of challenging proteins by ssNMR.

The type II secretion system (T2SS) is a multi-protein envelope-spanning assembly that 25 translocates a wide range of virulence factors, enzymes and effectors through the outer 26 membrane (OM) of many Gram-negative bacteria. Here, using electron cryotomography and 27 subtomogram averaging methods, we present the first in situ structure of an intact T2SS, imaged 28 within the human pathogen Legionella pneumophila. Although the T2SS has only limited 29 sequence and component homology with the evolutionarily-related Type IV pilus (T4P) system, 30 we show that their overall architectures are remarkably similar. Despite similarities, there are 31 also differences, including for instance that the T2SS-ATPase complex is usually present but 32 disengaged from the inner membrane, the T2SS has a much longer periplasmic vestibule, and it 33 has a short-lived flexible pseudopilus. Placing atomic models of the components into our ECT 34 map produced a complete architectural model of the intact T2SS that provides new insights into 35 the structure and function of its components, its position within the cell envelope, and the 36 interactions between its different subcomplexes. Overall, these structural results strongly support 37 the piston model for substrate extrusion. 38 39 40 All rights reserved. No reuse allowed without permission.

The self‐assembly of cellular macromolecular machines such as the bacterial flagellar motor requires the spatio‐temporal synchronization of gene expression with proper protein localization and association of dozens of protein components. In Salmonella and Escherichia coli, a sequential, outward assembly mechanism has been proposed for the flagellar motor starting from the inner membrane, with the addition of each new component stabilizing the previous one. However, very little is known about flagellar disassembly. Here, using electron cryo‐tomography and sub‐tomogram averaging of intact Legionella pneumophila, Pseudomonas aeruginosa, and Shewanella oneidensis cells, we study flagellar motor disassembly and assembly in situ. We first show that motor disassembly results in stable outer membrane‐embedded sub‐complexes. These sub‐complexes consist of the periplasmic embellished P‐ and L‐rings, and bend the membrane inward while it remains apparently sealed. Additionally, we also observe various intermediates of the assembly process including an inner‐membrane sub‐complex consisting of the C‐ring, MS‐ring, and export apparatus. Finally, we show that the L‐ring is responsible for reshaping the outer membrane, a crucial step in the flagellar assembly process.

Using a new Titan Krios stage equipped with a single-axis holder, we developed two methods to accelerate the collection of tilt-series. We demonstrate a continuous-tilting method that can record a tilt-series in seconds, but with loss of details finer than ~4 nm. We also demonstrate a fastincremental method that can record a tilt-series several-fold faster than current methods and with similar resolution. We characterize the utility of both methods in real biological electron cryotomography workflows. We identify opportunities for further improvements in hardware and software and speculate on the impact such advances could have on structural biology. Highlights Continuous-tilting: acquire tilt-series in seconds  Currently limited to an estimated 4 nm resolution  Fast-incremental: acquire tilt-series several-fold faster than current methods  Can produce high quality subtomogram average at a fraction of the time

1H-detection can greatly improve spectral sensitivity in biological solid-state NMR (ssNMR), thus allowing the study of larger and more complex proteins. However, the general requirement to perdeuterate proteins critically curtails the potential of 1H-detection by the loss of aliphatic side-chain protons, which are important probes for protein structure and function. Introduced herein is a labelling scheme for 1H-detected ssNMR, and it gives high quality spectra for both side-chain and backbone protons, and allows quantitative assignments and aids in probing interresidual contacts. Excellent 1H resolution in membrane proteins is obtained, the topology and dynamics of an ion channel were studied. This labelling scheme will open new avenues for the study of challenging proteins by ssNMR.