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In the last ten to 15 years, several developments have made solid-state nuclear magnetic resonance (NMR) spectroscopy a valuable technique in the field of structural biology. Stronger magnets (up to 28.2 T) have improved both sensitivity and resolution of the experiments, allowing the investigation of increasingly large and complex biomolecules. Fast magic-angle spinning (MAS) at frequencies exceeding 100 kHz is now capable of averaging the strong dipolar coupling between protons and other nuclei to an extent that enables the recording of well-resolved proton-detected spectra. The combination of sample rotation and simultaneous radio-frequency (rf) irradiation in solid-state NMR opens up the possibility of generating different effective Hamiltonians at different parts of an experiment, thus creating a huge and constantly growing variety of customised pulse sequences that are able to selectively correlate the nuclei of interest. Finally, the establishment of sedimentation as a sample preparation technique has made many soluble and non-crystallisable proteins accessible to solid-state NMR. In chapter 1, the theoretical foundations for solid-state NMR experiments are summarised. A particular focus is put on recent developments as well as aspects that are relevant for the ensuing parts of this work. The following chapters provide a biological background and summarise the available literature on the various biomolecular systems that were investigated in the projects presented in this work. These include the bacterial RNA helicase and acetyltransferase TmcA (chapter 2), the non-structural protein 5A of the hepatitis C virus (chapter 3), membrane proteins of the SARS coronavirus 2 (chapter 4) and reversible lysozyme fibrils (chapter 5). Chapter 6 encompasses the characterisation of the apo-state of TmcA. It starts with the work that had been done to establish this protein system for solid-state NMR studies, including cloning of the construct, the purification of the protein and sedimentation. An important aspect in this regards was also the resonance assignment, which was however limited by the size of the protein and accordingly spectral overlap. Still, it was possible to obtain heavy atom resonance assignments for 84 out of 671 residues, corresponding to about 13%. While the resonance assigment was done on ADP-bound TmcA, which yielded well-resolved carbon-detected spectra, sedimentation also allowed to analyse the non-crystallisable apo-state. Besides chemical-shift perturbations, which were located in the helicase portion of the protein, the resonances in spectra of apo-TmcA were considerably broader. Bulk relaxation measurements enabled to pinpoint this difference to a larger inhomogeneous linewidth, suggesting more structural heterogeneity in the absence of the nucleotide. This can also serve as an explanation why crystallisation attempts failed. Chapter 7 builds upon the work on sedimented TmcA presented in the preceding chapter to study the effect of the binding of various types of ligands to TmcA. First, the diamagnetic Mg(II) in the ATPase active site was substituted with paramagnetic Mn(II) and Co(II) to localise it within the protein, since the metal ion was absent in the model from X-ray crystallography. Paramagnetic relaxation enhancements (PRE) were used as distance restraints to triangulate the position of the metal ion, which was then confirmed by pseudo-contact shifts. The metal ion was found to be coordinated by conserved Walker motif residues and the beta-phosphate of the nucleotide, which is in agreement to structures of similar proteins. The PREs also allowed an estimation of the Mn(II) electron relaxation time. Secondly, TmcA was co-sedimented with various ATP analogues. This enabled to follow the ATP hydrolysis cycle from the perspective of the protein by carbon-detected experiments and from the perspective of the nucleotide by phosphorus-detected experiments. Finally, the binding of RNA and acetyl-coenzyme A was analysed, which were previously known to stimulate ATP hydrolysis. Interestingly, chemical-shift perturbations (CSPs) were not only observed close to the expected binding site, but also in the ATPase active site. While the solid-state NMR measurements on the bacterially expressed TmcA were carried out in 3.2 mm rotors, the next two chapters deal with proteins that were produced by cell-free synthesis using wheat-germ extract. The comparably low yields of this expression technique are better compatible with proton-detection in 0.7 mm rotors at fast MAS of 100 kHz. In chapter 8, continuing work on NS5A of the hepatitis C virus is presented. Our group has previously established a membrane-anchored NS5A construct reconstituted in liposomes for solid-state NMR. This was the basis for investigation of Daclatasvir binding, which is an approved drug against hepatitis C that targets NS5A. Spectra recorded at the highest available field of 28.2 T revealed that already in the absence of Daclatasvir, NS5A exists in two forms, with a major and minor species, potentially a dynamic equilibrium between monomer and dimer. Upon Daclatasvir binding, the previously minor form is stabilised to make up almost 100%. The distribution of chemical-shift changes on the crystal structure of a monomer suggests that the dimer is asymmetric in nature. Chapter 9 continues on the discussion of viral membrane proteins, this time of the SARS-CoV-2. These proteins proved to be challenging targets, with short T2' relaxation times and broad, unresolved spectra. Further analyses showed that the broadening predominantly occurs in the proton dimension, while carbon and nitrogen dimensions are better resolved. Broad spectra were also observed in the case of the reversible lysozyme fibrils, which are presented in chapter 10. Bulk relaxation measurements again shed light on the different contributions to the linewidth. It turned out that both the homogeneous and the inhomogeneous linewidths are exceptionally large. This suggests that the fibrils are characterised by extensive polymorphism, and the individual polymorphs are additionally highly dynamic. The different projects presented in this work constitute examples for the application of solid-state NMR spectroscopy to the study of complex biomolecules. In the conclusions, the opportunities and limitations of solid-state NMR as a tool in structural biology are discussed and it is contrasted with other techniques, in particular X-ray crystallography, but also cryo-electron microscopy and in-silico structure prediction.

Adhesive type 1 pili from uropathogenic Escherichia coli strains are filamentous, supramolecular protein complexes consisting of a short tip fibrillum and a long, helical rod formed by up to several thousand copies of the major pilus subunit FimA. Here, we reconstituted the entire type 1 pilus rod assembly reaction in vitro, using all constituent protein subunits in the presence of the assembly platform FimD, and identified the so-far uncharacterized subunit FimI as an irreversible assembly terminator. We provide a complete, quantitative model of pilus rod assembly kinetics based on the measured rate constants of FimD-catalyzed subunit incorporation. The model reliably predicts the length distribution of assembled pilus rods as a function of the ratio between FimI and the main pilus subunit FimA and is fully consistent with the length distribution of membrane-anchored pili assembled in vivo. The results show that the natural length distribution of adhesive pili formed via the chaperone-usher pathway results from a stochastic chain termination reaction., bioRxiv

Amidst the ongoing global climate crisis and the wake of the Russia-Ukraine war, renewable energy received a major boost in importance as independent and sustainable power supply. This report provides an in-depth analysis and valuation of Encavis, a major European independent power producer (IPP). This report constitutes the second part of a joint project and seamlessly ties in on the macroeconomic analysis and the historic financial analysis of Encavis. To enable a detailed valuation, key value drivers are identified and projected into the future, such as the expansion and performance of power plants and an own projection of future market prices based on expected LCOE values. The value of these projected developments is captured in a sum-of-the-part analysis for the existing plant portfolio, the pipeline projects, the option to repower retired plants, and the asset management segment. Based on the recent stock rally of Encavis in the last weeks we find that the market more than sufficiently prices in the expected growth ambitions and is slightly overvaluing, leading us to issue a SELL recommendation.

The subject matter of this work relates to micrometer-sized, droplet-based laser-based plasma light sources. The interaction between a high-power Nd:YAG laser nanosecond pulse and the target material is optimized for in-band spectral emission wavelength centered at 13.5 nm, also referred to as the extreme ultraviolet EUV wavelength range. The EUV-light emission from a laser-produced plasma (LPP) is used in the next-generation lithography processes for metrology and inspection tools during manufacturing and inspection, succeeding the 193nm-based inspection tools. For a light source to be economically viable, operating costs must be minimized while source reliability and EUV energy output stability must be maximized. A key challenge lies in optimizing the laser-target interaction at high operational frequencies, directly affecting the EUV-power stability and debris formation process. Furthermore, variations in the laser-target alignment can significantly affect the debris formation of the high kinetic ion emission, neutral particle emission, and the trajectory of liquid fragments, leading to contamination of sensitive optical components, such as the optical laser port or the EUV collecting optics. Droplet formation at low non-dimensional wavenumbers is prone to satellite droplet formation and perturbations in the drop-to-drop spacing. Therefore making the droplet generation process the most dominant source of laser-target misalignment and causing instabilities in the EUV power emission. A design modification of the in-house developed droplet generator focused on increasing the signal-to-noise ratio (SNR) of the carrier and modulation frequency components of the amplitude modulate actuation signal. The piezoelectric piston actuation system was designed for stable droplet generation at 33 kHz, and the SNR was confirmed to be >28 for maximum amplitude operation and >103 for optimal droplet formation conditions, which was confirmed by laser Doppler vibrometer measurements. Increasing the cartridge's pressure rating (>100 bar) and the maximum power of the piezoelectric piston actuation system-induced pressure perturbation by 35 compared to the previous version increased the relative droplet spacing to >14. Hence, the operational range of the droplet generator is increased, providing continuous spherical targets with a diameter range of 42μm to 84 μm and droplet velocity ranging from 7ms-1 to 30 ms-1. The increased droplet velocity is required to increase the interdroplet spacing and reduce the effect of high debris mitigation gas flow rates on the droplet trajectory. The droplet stability performance assessment is conducted by the upgraded tracking system with a reduced time resolution of 10 ns for measuring the interdroplet temporal spacing. The droplet camera system assesses the low-frequency two-dimensional target shape directly before the laser pulse interacts with the target surface, which can resolve the droplet diameter or shape with a resolution of 22 hours is achieved with pulse-to-pulse stability of 3σ=4.1% and an averaged EUV power stability over 10 milliseconds of

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium’s collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form., This work was supported by Goethe University (Corona funds), the DFG-funded CRC: “Molecular Principles of RNA-Based Regulation,” DFG infrastructure funds (project numbers: 277478796, 277479031, 392682309, 452632086, 70653611), the state of Hesse (BMRZ), the Fondazione CR Firenze (CERM), and the IWB-EFRE-program 20007375. This project has received funding from the European Union’s Horizon 2020 research and innovation program under Grant Agreement No. 871037. AS is supported by DFG Grant SCHL 2062/2-1 and by the JQYA at Goethe through project number 2019/AS01. Work in the lab of KV was supported by a CoRE grant from the University of New Hampshire. The FLI is a member of the Leibniz Association (WGL) and financially supported by the Federal Government of Germany and the State of Thuringia. Work in the lab of RM was supported by NIH (2R01EY021514) and NSF (DMR-2002837). BN-B was supported by theNSF GRFP.MCwas supported byNIH (R25 GM055246 MBRS IMSD), and MS-P was supported by the HHMI Gilliam Fellowship. Work in the labs of KJ and KT was supported by Latvian Council of Science Grant No. VPP-COVID 2020/1-0014. Work in the UPAT’s lab was supported by the INSPIRED (MIS 5002550) project, which is implemented under the Action “Reinforcement of the Research and Innovation Infrastructure,” funded by the Operational Program “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014–2020) and cofinanced by Greece and the EU (European Regional Development Fund) and the FP7 REGPOT CT-2011- 285950–“SEE-DRUG” project (purchase of UPAT’s 700MHz NMR equipment). Work in the CM-G lab was supported by the Helmholtz society. Work in the lab of ABö was supported by the CNRS, the French National Research Agency (ANR, NMRSCoV2- ORF8), the Fondation de la Recherche Médicale (FRM, NMR-SCoV2-ORF8), and the IR-RMN-THC Fr3050 CNRS. Work in the lab of BM was supported by the Swiss National Science Foundation (Grant number 200020_188711), the Günthard Stiftung für Physikalische Chemie, and the ETH Zurich. Work in the labs of ABö and BM was supported by a common grant from SNF (grant 31CA30_196256). This work was supported by the ETHZurich, the grant ETH40 18 1, and the grant Krebsliga KFS 4903 08 2019. Work in the lab of the IBS Grenoble was supported by the Agence Nationale de Recherche (France) RA-COVID SARS2NUCLEOPROTEIN and European Research Council Advanced Grant DynamicAssemblies. Work in the CA lab was supported by Patto per il Sud della Regione Siciliana–CheMISt grant (CUP G77B17000110001). Part of this work used the platforms of the Grenoble Instruct-ERIC center (ISBG; UMS 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10-INBS-05-02) and GRAL, financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE- 0003). Work at the UW-Madison was supported by grant numbers NSF MCB2031269 and NIH/NIAID AI123498. MM is a Ramón y Cajal Fellow of the Spanish AEI-Ministry of Science and Innovation (RYC2019-026574-I), and a “La Caixa” Foundation (ID 100010434) Junior Leader Fellow (LCR/BQ/PR19/11700003). Funded by project COV20/00764 fromthe Carlos III Institute of Health and the SpanishMinistry of Science and Innovation to MMand DVL. VDJ was supported by the Boehringer Ingelheim Fonds. Part of this work used the resources of the Italian Center of Instruct-ERIC at the CERM/ CIRMMP infrastructure, supported by the Italian Ministry for University and Research (FOE funding). CF was supported by the Stiftung Polytechnische Gesellschaft. Work in the lab of JH was supported by NSF (RAPID 2030601) and NIH (R01GM123249).

The Hepatitis C virus nonstructural protein 5A (NS5A) is a membrane-associated protein involved in multiple steps of the viral life cycle. Direct-acting antivirals (DAAs) targeting NS5A are a cornerstone of antiviral therapy, but the mode-of-action of these drugs is poorly understood. This is due to the lack of information on the membrane-bound NS5A structure. Herein, we present the structural model of an NS5A AH-linker-D1 protein reconstituted as proteoliposomes. We use highly sensitive proton-detected solid-state NMR methods suitable to study samples generated through synthetic biology approaches. Spectra analyses disclose that both the AH membrane anchor and the linker are highly flexible. Paramagnetic relaxation enhancements (PRE) reveal that the dimer organization in lipids requires a new type of NS5A self-interaction not reflected in previous crystal structures. In conclusion, we provide the first characterization of NS5A AH-linker-D1 in a lipidic environment shedding light onto the mode-of-action of clinically used NS5A inhibitors. ISSN:1433-7851 ISSN:1521-3773 ISSN:0570-0833

Over the last decades, various predictors have proven relevant for job performance [e.g., general mental ability (GMA), broad personality traits, such as the Big Five]. However, prediction of job performance is far from perfect, and further potentially relevant predictors need to be investigated. Narrower personality traits, such as individuals' character strengths, have emerged as meaningfully related to different aspects of job performance. However, it is still unclear whether character strengths can explain additional variance in job performance over and above already known powerful predictors. Consequently, the present study aimed at (1) examining the incremental validity of character strengths as predictors of job performance beyond GMA and/or the Big Five traits and (2) identifying the most important predictors of job performance out of the 24 character strengths, GMA, and the Big Five. Job performance was operationalized with multidimensional measures of both productive and counterproductive work behavior. A sample of 169 employees from different occupations completed web-based self-assessments on character strengths, GMA, and the Big Five. Additionally, the employees' supervisors provided web-based ratings of their job performance. Results showed that character strengths incrementally predicted job performance beyond GMA, the Big Five, or GMA plus the Big Five; explained variance increased up to 54.8, 43.1, and 38.4%, respectively, depending on the dimension of job performance. Exploratory relative weight analyses revealed that for each of the dimensions of job performance, at least one character strength explained a numerically higher amount of variance than GMA and the Big Five, except for individual task proactivity, where GMA exhibited the numerically highest amount of explained variance. The present study shows that character strengths are relevant predictors of job performance in addition to GMA and other conceptualizations of personality (i.e., the Big Five). This also highlights the role of socio-emotional skills, such as character strengths, for the understanding of performance outcomes above and beyond cognitive ability.