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A new high-pressure single-crystal diffraction setup has been designed and implemented at the Australian Synchrotron for collecting molecular and protein crystal structures. The setup incorporates a modified micro-Merrill-Bassett cell and holder designed specifically to fit onto the horizontal air-bearing goniometer, allowing high-pressure diffraction measurements to be collected with little to no modification of the beamline setup compared with ambient data collections. Compression data for the amino acid, l-threonine, and the protein, hen egg-white lysozyme, were collected, showcasing the capabilities of the setup. [ABSTRACT FROM AUTHOR]

RNA-binding proteins of the DBHS (Drosophila Behavior Human Splicing) family, NONO, SFPQ, and PSPC1 have numerous roles in genome stability and transcriptional and posttranscriptional regulation. Critical to DBHS activity is their recruitment to distinct subnuclear locations, for example, paraspeckle condensates, where DBHS proteins bind to the long noncoding RNA NEAT1 in the first essential step in paraspeckle formation. To carry out their diverse roles, DBHS proteins form homodimers and heterodimers, but how this dimerization influences DBHS localization and function is unknown. Here, we present an inducible GFP-NONO stable cell line and use it for live-cell 3D-structured illumination microscopy, revealing paraspeckles with dynamic, twisted elongated structures. Using siRNA knockdowns, we show these labeled paraspeckles consist of GFP-NONO/endogenous SFPQ dimers and that GFP-NONO localization to paraspeckles depends on endogenous SFPQ. Using purified proteins, we confirm that partner swapping between NONO and SFPQ occurs readily in vitro. Crystallographic analysis of the NONOSFPQ heterodimer reveals conformational differences to the other DBHS dimer structures, which may contribute to partner preference, RNA specificity, and subnuclear localization. Thus overall, our study suggests heterodimer partner availability is crucial for NONO subnuclear distribution and helps explain the complexity of both DBHS protein and paraspeckle dynamics through imaging and structural approaches. [ABSTRACT FROM AUTHOR]

Chymotrypsin is a protease that is commonly used as a standard for protein crystallization and as a model system for studying serine proteases. Unliganded bovine α-chymotrypsin was crystallized at neutral pH using ammonium sulphate as the precipitant, resulting in crystals that conform to P65 symmetry with unit cell parameters that have not been reported previously. Inspection of crystallographic interfaces revealed that the major interface between any two molecules in the crystal lattice represents the interface of the biological dimer, as previously observed for crystals of unliganded α-chymotrypsin grown at low pH in space group P21. [ABSTRACT FROM AUTHOR]