Eukaryotic ribosome biogenesis depends on several hundred assembly factors to produce functional 40S and 60S ribosomal subunits. The final phase of 60S subunit biogenesis is cytoplasmic maturation, which includes the proofreading of functional centers of the 60S subunit and the release of several ribosome biogenesis factors. We report the cryo-electron microscopy (cryo-EM) structure of the yeast 60S subunit in complex with the biogenesis factors Rei1, Arx1, and Alb1 at 3.4 Å resolution. In addition to the network of interactions formed by Alb1, the structure reveals a mechanism for ensuring the integrity of the ribosomal polypeptide exit tunnel. Arx1 probes the entire set of inner-ring proteins surrounding the tunnel exit, and the C terminus of Rei1 is deeply inserted into the ribosomal tunnel, where it forms specific contacts along almost its entire length. We provide genetic and biochemical evidence that failure to insert the C terminus of Rei1 precludes subsequent steps of 60S maturation.

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The structure of Bacillus amyloliquefaciens ribonuclease (barnase), an extracellular 110-residue enzyme initially solved at 2.0 A resolution, has been refined at 1.5 A using synchrotron radiation and an imaging-plate scanner. Refinement with anisotropic atomic displacement parameters resulted in increased accuracy of the structure. The final model has a crystallographic R factor of 11.5% and an Rfree of 17.4%. The three independent molecules in the asymmetric unit, referred to as A, B and C, allowed detailed analysis of this final model and meaningful comparison with structures of barnase complexed either with nucleotide inhibitors or with its natural intracellular inhibitor, barstar. The analysis of the overall solvent structure revealed a similar number of water molecules associated with each barnase molecule; among these were 16 equivalent buried solvent molecules, the locations of which are discussed in detail and classified on the basis of their structural role. The importance of the water molecules' contribution to the barnase-barstar interaction is also highlighted. The high accuracy of the present analysis revealed the presence of a Zn2+ ion mediating the contacts between pairs of symmetry-related A, B or C molecules; such an ion had previously only been identified for pairs of C molecules.

PDB Reference: barnase cross-linked by glutaraldehyde, 3kch.In addition to the common use of glutaraldehyde to nonspecifically cross-link protein crystals through lysine residues disposed on the surface of the protein, the use of gentle vapour diffusion of glutaraldehyde offers a convenient way to limit polymerization and to allow slow diffusion throughout the crystal. In the case of trimeric barnase crystals, a specific cross-link was observed between an lysine side chain and an arginine side chain that were spatially disposed at the ideal distance on the protein surface in the three monomers. Here, the direct observation of a specific Lys-Arg cross-link site is reported and a mechanism is proposed for the reaction.

Effects of ionic (Hypaque-76) and nonionic (Isovue-370 and Omnipaque-350) contrast media on oxyhemoglobin dissociation of normal human red blood cells were evaluated. In series 1, 4-mL venous blood samples were obtained from 15 normal human volunteers. One blood sample served as control, and 1 mL of either of the three contrast media was added in vitro to the other 4-mL blood samples. P50 values were estimated from the linear portion of the oxyhemoglobin dissociation curve obtained by tonometry. Determinations of P50 were performed at either pH 7.4 or 7.2. At pH 7.4, P50 in the absence of contrast media was 26.3 +/- 0.4 mm Hg (mean +/- SEM). The contrast media caused comparable decreases in P50 from this value (Hypaque-76, 20.0 +/- 0.5 mm Hg; Omnipaque-350, 21.6 +/- 0.4 mm Hg; Isovue-370, 20.7 +/- 0.4 mm Hg). Reducing pH to 7.2 in the absence of contrast media increased P50 to 33.3 +/- 1.0 mm Hg, evidence of the Bohr effect. The presence of contrast media either completely abolished (Hypaque-76 and Omnipaque-350) or markedly attenuated (Isovue-370) this effect. In series 2 (five patients), blood samples were withdrawn from the external iliac artery during injection of Isovue-370 (60-78 mL) into the proximal abdominal aorta to evaluate peripheral vascular disease. Measurement of P50 of these samples yielded findings consistent with those of series 1. The present findings demonstrate that both ionic and nonionic contrast media increase the affinity of hemoglobin for oxygen and, therefore, that they may inhibit oxygen delivery to body tissues.

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One of the major families of membrane proteins found in prokaryote genome corresponds to the transporters. Among them, the resistance-nodulation-cell division (RND) transporters are highly studied, as being responsible for one of the most problematic mechanisms used by bacteria to resist to antibiotics, i.e., the active efflux of drugs. In Gram-negative bacteria, these proteins are inserted in the inner membrane and form a tripartite assembly with an outer membrane factor and a periplasmic linker in order to cross the two membranes to expulse molecules outside of the cell. A lot of information has been collected to understand the functional mechanism of these pumps, especially with AcrAB-TolC from Escherichia coli, but one missing piece from all the suggested models is the role of peptidoglycan in the assembly. Here, by pull-down experiments with purified peptidoglycans, we precise the MexAB-OprM interaction with the peptidoglycan from Escherichia coli and Pseudomonas aeruginosa, highlighting a role of the peptidoglycan in stabilizing the MexA-OprM complex and also differences between the two Gram-negative bacteria peptidoglycans.

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