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Tau is an intrinsically disordered protein that binds and stabilizes axonal microtubules (MTs) in neurons of the central nervous system. The binding of Tau to MTs is mediated by its repeat domain and flanking proline-rich domains. The positively charged (basic) C-terminal half of Tau also mediates the assembly Tau into fibrillar aggregates in Alzheimer's disease (AD) and tauopathy brains. In recent years, another assembly form of Tau has been identified: Tau can undergo liquid-liquid phase separation (LLPS), which leads to its condensation into liquid-dense phases, either by complex coacervation with polyanions like heparin or RNA or through 'self-coacervation' at high Tau concentrations. Condensation of Tau in the absence of polyanions can be enhanced by the presence of molecular crowding agents in a dilute Tau solution. In vitro experiments using recombinant purified Tau are helpful to study the physicochemical determinants of Tau LLPS, which can then be extrapolated into the cellular context. Tau condensation is a new aspect of Tau biology that may play a role for the initiation of Tau aggregation, but also for its physiological function(s), for example, the binding to microtubules. Here we describe how to study the condensation of Tau in vitro using light microscopy, including fluorescence recovery after photobleaching (FRAP), to assess the shape and molecular diffusion in the condensates, a proxy for the degree of condensate percolation. We also describe turbidity measurements of condensate-containing solutions to assess the overall amount of LLPS and time-resolved dynamic light scattering (trDLS) to study the formation and size of Tau condensates.

Additional file 1: Figure S1: Genomic DNA from Fusarium metavorans FW16.1 (DSM105788) was isolated using the CTAB method and 5 μL was mixed with 6 × loading buffer (0.25% (w/v) xylene cyanol, 0.25% (w/v) bromophenol blue, 30% (v/v) glycerol) and separated by 0.8% (w/v) agarose gel electrophoresis in Tris–borate EDTA (TBE) buffer at 80 V for 60 min, with the GeneRuler 1 kb Plus DNA Ladder (Thermo Fisher Scientific) as a marker. The DNA was stained with 1% ethidium bromide for 15 min and observed on a UV transilluminator (SynGene Genius, BioImaging System). Figure S2: Specific CMCase activity of the supernatants against high-viscosity CMC over time in YPD medium. Figure S3: Specific CMCase activity of the supernatants using different synthetic nutrient sources. Figure S4: Enzymatic activities for polygalacturonase (A), laminarinase (B), CMCase (C) and xylanase (D). Table S1: CMCase activity of 48 fungal strains. Table S2: CAZyme analysis of fungal isolate FW16.1 and other fungal species. The coding regions were compared with the CAZyme database (Cantarel et al. 2009; Lombard et al. 2014). Table S3: Proteins of the fungal isolate FW16.1 induced on different synthetic and artificial cellulose and biomass substrates (maize leaves (MZ) or sugar cane bagasse (SCB)). The proteins were separated by SDS-PAGE followed by in-gel tryptic digestion and LC–MS/MS. The accession number, description, coverage (%), number of peptides (# peptides), peptide-to-spectrum matches (# PSMs), molecular weight in kDa (MW [kDa]), the calculated isoelectric point (calc. pI), Score Sequest HT and number of Peptides Sequest HT (# Peptides Sequest HT) were compared with the automated translation of the genome of the fungal isolate Fusarium metavorans DSM105788. BLASTP annotations and cellular functions are also shown. Table S4: Functional prediction of the CAZymes found on different synthetic and artificial cellulose and biomass substrates. Table S5: Quantity of protein applied in the saturation curve experiments.

The new European X-ray Free-Electron Laser is the first X-ray free-electron laser capable of delivering X-ray pulses with a megahertz inter-pulse spacing, more than four orders of magnitude higher than previously possible. However, to date, it has been unclear whether it would indeed be possible to measure high-quality diffraction data at megahertz pulse repetition rates. Here, we show that high-quality structures can indeed be obtained using currently available operating conditions at the European XFEL. We present two complete data sets, one from the well-known model system lysozyme and the other from a so far unknown complex of a β-lactamase from K. pneumoniae involved in antibiotic resistance. This result opens up megahertz serial femtosecond crystallography (SFX) as a tool for reliable structure determination, substrate screening and the efficient measurement of the evolution and dynamics of molecular structures using megahertz repetition rate pulses available at this new class of X-ray laser source., The new European X-Ray Free-Electron Laser (EuXFEL) is the first XFEL that generates X-ray pulses with a megahertz inter-pulse spacing. Here the authors demonstrate that high-quality and damage-free protein structures can be obtained with the currently available 1.1 MHz repetition rate pulses using lysozyme as a test case and furthermore present a β-lactamase structure.

Nucleic acids research / Special publication 38(19), 6729 - 6736 (2010). doi:10.1093/nar/gkq505, 'Locked nucleic acids' (LNAs) are known to introduce enhanced bio- and thermostability into natural nucleic acids rendering them powerful tools for diagnostic and therapeutic applications. We present the 1.9 Å X-ray structure of an 'all LNA' duplex containing exclusively modified β-D-2'-O-4'C-methylene ribofuranose nucleotides. The helix illustrates a new type of nucleic acid geometry that contributes to the understanding of the enhanced thermostability of LNA duplexes. A notable decrease of several local and overall helical parameters like twist, roll and propeller twist influence the structure of the LNA helix and result in a widening of the major groove, a decrease in helical winding and an enlarged helical pitch. A detailed structural comparison to the previously solved RNA crystal structure with the corresponding base pair sequence underlines the differences in conformation. The surrounding water network of the RNA and the LNA helix shows a similar hydration pattern., Published by Information Retrieval Ltd., London