A simple pressure assisted method for MicroED specimen preparation

Micro-crystal electron diffraction (MicroED) has shown great potential for structure determination of macromolecular crystals too small for X-ray diffraction. However, specimen preparation remains a major bottleneck. Here, we report a simple method for preparing MicroED specimens, named Preassis, in which excess liquid is removed through an EM grid with the assistance of pressure. We show the ice thicknesses can be controlled by tuning the pressure in combination with EM grids with appropriate carbon hole sizes. Importantly, Preassis can handle a wide range of protein crystals grown in various buffer conditions including those with high viscosity, as well as samples with low crystal concentrations. Preassis is a simple and universal method for MicroED specimen preparation, and will significantly broaden the applications of MicroED

Current status and future opportunities for serial crystallography at MAX IV Laboratory

Over the last decade, serial crystallography, a method to collect complete diffraction datasets from a large number of microcrystals delivered and exposed to an X-ray beam in random orientations at room temperature, has been successfully implemented at X-ray free-electron lasers and synchrotron radiation facility beamlines. This development relies on a growing variety of sample presentation methods, including different fixed target supports, injection methods using gas-dynamic virtual-nozzle injectors and high-viscosity extrusion injectors, and acoustic levitation of droplets, each with unique requirements. In comparison with X-ray free-electron lasers, increased beam time availability makes synchrotron facilities very attractive to perform serial synchrotron X-ray crystallography (SSX) experiments. Within this work, the possibilities to perform SSX at BioMAX, the first macromolecular crystallography beamline at MAX IV Laboratory in Lund, Sweden, are described, together with case studies from the SSX user program: an implementation of a high-viscosity extrusion injector to perform room temperature serial crystallography at BioMAX using two solid supports - silicon nitride membranes (Silson, UK) and XtalTool (Jena Bioscience, Germany). Future perspectives for the dedicated serial crystallography beamline MicroMAX at MAX IV Laboratory, which will provide parallel and intense micrometre-sized X-ray beams, are discussed

Comparative structural analysis provides new insights into the function of R2 like ligand binding oxidase

R2 like ligand binding oxidase R2lox is a ferritin like protein that harbours a heterodinuclear manganese iron active site. Although R2lox function is yet to be established, the enzyme binds a fatty acid ligand coordinating the metal centre and catalyses the formation of a tyrosine valine ether cross link in the protein scaffold upon O2 activation. Here, we characterized the ligands copurified with R2lox by mass spectrometry based metabolomics. Moreover, we present the crystal structures of two new homologs of R2lox, from Saccharopolyspora amp; 8201;erythraea and Sulfolobus amp; 8201;acidocaldarius, at 1.38 amp; 8201; and 2.26 amp; 8201; resolution, respectively, providing the highest resolution structure for R2lox, as well as new insights into putative mechanisms regulating the function of the enzym