Nuclear transport: what a kary-on!

AbstractCompartmentalisation in eukaryotic cells presents special problems in macromolecular transport. Here we use the recently determined X-ray structures of a number of components of the nuclear transport machinery as a framework to review current understanding of this fundamental biological process

Structures of complexes formed by H5 influenza hemagglutinin with a potent broadly neutralizing human monoclonal antibody.

H5N1 avian influenza viruses remain a threat to public health mainly because they can cause severe infections in humans. These viruses are widespread in birds, and they vary in antigenicity forming three major clades and numerous antigenic variants. The most important features of the human monoclonal antibody FLD194 studied here are its broad specificity for all major clades of H5 influenza HAs, its high affinity, and its ability to block virus infection, in vitro and in vivo. As a consequence, this antibody may be suitable for anti-H5 therapy and as a component of stockpiles, together with other antiviral agents, for health authorities to use if an appropriate vaccine was not available. Our mutation and structural analyses indicate that the antibody recognizes a relatively conserved site near the membrane distal tip of HA, near to, but distinct from, the receptor-binding site. Our analyses also suggest that the mechanism of infectivity neutralization involves prevention of receptor recognition as a result of steric hindrance by the Fc part of the antibody. Structural analyses by EM indicate that three Fab fragments are bound to each HA trimer. The structure revealed by X-ray crystallography is of an HA monomer bound by one Fab. The monomer has some similarities to HA in the fusion pH conformation, and the monomer's formation, which results from the presence of isopropanol in the crystallization solvent, contributes to considerations of the process of change in conformation required for membrane fusion

The Structural Basis for 14-3-3:Phosphopeptide Binding Specificity

AbstractThe 14-3-3 family of proteins mediates signal transduction by binding to phosphoserine-containing proteins. Using phosphoserine-oriented peptide libraries to probe all mammalian and yeast 14-3-3s, we identified two different binding motifs, RSXpSXP and RXY/FXpSXP, present in nearly all known 14-3-3 binding proteins. The crystal structure of 14-3-3ζ complexed with the phosphoserine motif in polyoma middle-T was determined to 2.6 Å resolution. The bound peptide is in an extended conformation, with a tight turn created by the pS +2 Pro in a cis conformation. Sites of peptide–protein interaction in the complex rationalize the peptide library results. Finally, we show that the 14-3-3 dimer binds tightly to single molecules containing tandem repeats of phosphoserine motifs, implicating bidentate association as a signaling mechanism with molecules such as Raf, BAD, and Cbl

Influenza hemagglutinin membrane anchor

Viruses with membranes fuse them with cellular membranes, to transfer their genomes into cells at the beginning of infection. For Influenza virus, the membrane glycoprotein involved in fusion is the hemagglutinin (HA), the 3D structure of which is known from X-ray crystallographic studies. The soluble ectodomain fragments used in these studies lacked the “membrane anchor” portion of the molecule. Since this region has a role in membrane fusion, we have determined its structure by analyzing the intact, full-length molecule in a detergent micelle, using cryo-EM. We have also compared the structures of full-length HA−detergent micelles with full-length HA−Fab complex detergent micelles, to describe an infectivity-neutralizing monoclonal Fab that binds near the ectodomain membrane anchor junction. We determine a high- resolution HA structure which compares favorably in detail with the structure of the ectodomain seen by X-ray crystallography; we detect, clearly, all five carbohydrate side chains of HA; and we find that the ectodomain is joined to the membrane anchor by flexible, eight-residue-long, linkers. The linkers extend into the detergent micelle to join a central triple-helical structure that is a major component of the membrane anchor

A Simple Air-Stream Clay Separator

A simple, air-stream clay separator was devised to separate the 30 mm and smaller fraction from relatively large amounts of dry clay. The separator can process approximately 36 kg of clay in six hours with an average product yield of about 800 g, depending upon the initial particle size distribution of the clay. By increasing the length of the separator the design can be altered to separate an even smaller size fraction. The air-stream separator could be used as a simple and expedient preconcentration step prior to final separation in a typical laboratorymodel, low-volume hydrocyclone, or for separation of the clay fraction from dry soil. The air separator is based on the principle that smaller, lighter clay particles travel farther than larger, heavier particles before settling out of the air stream. Of course, particle clumps act as individual particles. Therefore, unflocculated, dry clay gives the best yield in the air separator

Antibody-mediated disruption of the SARS-CoV-2 spike glycoprotein

Here, the authors characterise the binding and neutralisation properties of CR3022, a neutralising Ab isolated from a convalescent SARS patient, against SARS-CoV-2 spike trimers and show using Cryo-EM the disruption of SARS-CoV-2 spike by CR3022 Fab

MgF(3)(-) as a transition state analog of phosphoryl transfer

AbstractThe formation of complexes between small G proteins and certain of their effectors can be facilitated by aluminum fluorides. Solution studies suggest that magnesium may be able to replace aluminum in such complexes. We have determined the crystal structure of RhoA.GDP bound to RhoGAP in the presence of Mg2+ and F− but without Al3+. The metallofluoride adopts a trigonal planar arrangement instead of the square planar structure of AlF4−. We have confirmed that these crystals contain magnesium and not aluminum by proton-induced X-ray emission spectroscopy. The structure adopted by GDP.MgF− possesses the stereochemistry and approximate charge expected for the transition state. We suggest that MgF3− may be the reagent of choice for studying phosphoryl transfer reactions