Structures from intact myofibrils reveal mechanism of thin filament regulation through nebulin.

In skeletal muscle, nebulin stabilizes and regulates the length of thin filaments, but the underlying mechanism remains nebulous. In this work, we used cryo-electron tomography and subtomogram averaging to reveal structures of native nebulin bound to thin filaments within intact sarcomeres. This in situ reconstruction provided high-resolution details of the interaction between nebulin and actin, demonstrating the stabilizing role of nebulin. Myosin bound to the thin filaments exhibited different conformations of the neck domain, highlighting its inherent structural variability in muscle. Unexpectedly, nebulin did not interact with myosin or tropomyosin, but it did interact with a troponin T linker through two potential binding motifs on nebulin, explaining its regulatory role. Our structures support the role of nebulin as a thin filament "molecular ruler" and provide a molecular basis for studying nemaline myopathies.

Recombination mechanisms of luminescence type gas sensors.

Luminescence type gas sensors based on organic luminophores are characterized by an excellent signal stability over the luminophores' lifetime. Even though the sensing material is prone to degradation due to photobleaching, evaluation of the dynamic optical response allows to minimize aging induced drift effects of the luminophore and the optoelectronic components. The gas dependent luminophore decay time is mostly independent of the excitation intensity, which is attributed to the monomolecular recombination in many organic luminophores. To improve the overall sensor lifetime and to utilize this luminescence based sensor concept for long-term sensing applications, new luminophores are needed. Potential candidates are inorganic, semiconducting materials, which, however, show more complex recombination behaviour than the organic luminophores mentioned before, involving also bimolecular recombination. In the scope of this work, the differences of mono- and bimolecular recombination are discussed by the use of a simple statistical model. The theoretical aspects are furthermore confirmed by dynamic luminescence measurements on manganese doped zinc sulfide (ZnS). The semiconducting ZnS is an oxygen sensitive luminophore, which shows both, mono- and bimolecular recombination, depending on the excitation energy.

Two particle-picking procedures for filamentous proteins: SPHIRE-crYOLO filament mode and SPHIRE-STRIPER.

Structure determination of filamentous molecular complexes involves the selection of filaments from cryo-EM micrographs. The automatic selection of helical specimens is particularly difficult, and thus many challenging samples with issues such as contamination or aggregation are still manually picked. Here, two approaches for selecting filamentous complexes are presented: one uses a trained deep neural network to identify the filaments and is integrated in SPHIRE-crYOLO, while the other, called SPHIRE-STRIPER, is based on a classical line-detection approach. The advantage of the crYOLO-based procedure is that it performs accurately on very challenging data sets and selects filaments with high accuracy. Although STRIPER is less precise, the user benefits from less intervention, since in contrast to crYOLO, STRIPER does not require training. The performance of both procedures on Tobacco mosaic virus and filamentous F-actin data sets is described to demonstrate the robustness of each method.

Cellulose Nanocrystal-Templated Tin Dioxide Thin Films for Gas Sensing.

Porous tin dioxide is an important low-cost semiconductor applied in electronics, gas sensors, and biosensors. Here, we present a versatile template-assisted synthesis of nanostructured tin dioxide thin films using cellulose nanocrystals (CNCs). We demonstrate that the structural features of CNC-templated tin dioxide films strongly depend on the precursor composition. The precursor properties were studied by using low-temperature nuclear magnetic resonance spectroscopy of tin tetrachloride in solution. We demonstrate that it is possible to optimize the precursor conditions to obtain homogeneous precursor mixtures and therefore highly porous thin films with pore dimensions in the range of 10-20 nm (ABET = 46-64 m2 g-1, measured on powder). Finally, by exploiting the high surface area of the material, we developed a resistive gas sensor based on CNC-templated tin dioxide. The sensor shows high sensitivity to carbon monoxide (CO) in ppm concentrations and low cross-sensitivity to humidity. Most importantly, the sensing kinetics are remarkably fast; both the response to the analyte gas and the signal decay after gas exposure occur within a few seconds, faster than in standard SnO2-based CO sensors. This is attributed to the high gas accessibility of the very thin porous film.

SPHIRE-crYOLO is a fast and accurate fully automated particle picker for cryo-EM.

Selecting particles from digital micrographs is an essential step in single-particle electron cryomicroscopy (cryo-EM). As manual selection of complete datasets-typically comprising thousands of particles-is a tedious and time-consuming process, numerous automatic particle pickers have been developed. However, non-ideal datasets pose a challenge to particle picking. Here we present the particle picking software crYOLO which is based on the deep-learning object detection system You Only Look Once (YOLO). After training the network with 200-2500 particles per dataset it automatically recognizes particles with high recall and precision while reaching a speed of up to five micrographs per second. Further, we present a general crYOLO network able to pick from previously unseen datasets, allowing for completely automated on-the-fly cryo-EM data preprocessing during data acquisition. crYOLO is available as a standalone program under http://sphire.mpg.de/ and is distributed as part of the image processing workflow in SPHIRE.

Structural transitions of F-actin upon ATP hydrolysis at near-atomic resolution revealed by cryo-EM.

The function of actin is coupled to the nucleotide bound to its active site. ATP hydrolysis is activated during polymerization; a delay between hydrolysis and inorganic phosphate (Pi) release results in a gradient of ATP, ADP-Pi and ADP along actin filaments (F-actin). Actin-binding proteins can recognize F-actin's nucleotide state, using it as a local 'age' tag. The underlying mechanism is complex and poorly understood. Here we report six high-resolution cryo-EM structures of F-actin from rabbit skeletal muscle in different nucleotide states. The structures reveal that actin polymerization repositions the proposed catalytic base, His161, closer to the γ-phosphate. Nucleotide hydrolysis and Pi release modulate the conformational ensemble at the periphery of the filament, thus resulting in open and closed states, which can be sensed by coronin-1B. The drug-like toxin jasplakinolide locks F-actin in an open state. Our results demonstrate in detail how ATP hydrolysis links to F-actin's conformational dynamics and protein interaction.

Tungsten Oxide Photonic Crystals as Optical Transducer for Gas Sensing.

Some metal oxide semiconductors, such as tungsten trioxide or tin dioxide, are well-known as resistive transducers for gas sensing and offer high sensitivities down to the part per billion level. Electrical signal read-out, however, limits the information obtained on the electronic properties of metal oxides to a certain frequency range and its application because of the required electrical contacts. Therefore, a novel approach for building an optical transducer for gas reactions utilizing metal oxide photonic crystals is presented here. By the rational design of the structure and composition it is possible to synthesize a functional material which allows one to obtain insight into its electronic properties in the optical frequency range with simple experimental measures. The concept is demonstrated by tungsten trioxide inverse opal structure as optical transducer material for hydrogen sensing. The sensing behavior is analyzed in a temperature range from room temperature to 500 °C and in a wide hydrogen concentration range (3000 ppm to 10%). The sensing mechanism is mainly the refractive index change resulting from hydrogen intercalation in tungsten trioxide, but the back reaction has also impact on the optical properties of this system. Detailed chemical reaction studies provide suggestions for specific sensing conditions.

Mesoporous materials as gas sensors.

Ordered mesoporous materials have great potential in the field of gas sensing. Today various template-assisted synthesis methods facilitate the preparation of silica (SiO2) as well as numerous metal oxides with well-defined, uniform and regular pore systems. The unique nanostructural properties of such materials are particularly useful for their application as active layers in gas sensors based on various operating principles, such as capacitive, resistive, or optical sensing. This review summarizes the basic aspects of materials synthesis, discusses some structural properties relevant in gas sensing, and gives an overview of the literature on ordered mesoporous gas sensors.

Photoreduction of mesoporous In2O3: mechanistic model and utility in gas sensing.

A model is proposed for the drop in electronic resistance of n-type semiconducting indium oxide (In(2)O(3)) upon illumination with light (350 nm, 3.5 eV) as well as for the (light-enhanced) sensitivity of In(2)O(3) to oxidizing gases. Essential features of the model are photoreduction and a rate-limiting oxygen-diffusion step. Ordered, mesoporous In(2)O(3) with a high specific surface area serves as a versatile system for experimental studies. Analytical techniques comprise conductivity measurements under a controlled atmosphere (synthetic air, pure N(2)) and temperature-resolved in-situ Fourier transform infrared (FTIR) spectroscopy. IR measurements reveal that oxygen vacancies form a donor level 0.18 eV below the conduction band.