f Nonstructural proteins 7 and 8 of severe acute respiratory syndrome coronavirus (SARS-CoV) have previously been shown by X-ray crystallography to form an 8:8 hexadecamer. In addition, it has been demonstrated that N-terminally His 6 -tagged SARSCoV Nsp8 is a primase able to synthesize RNA oligonucleotides with a length of up to 6 nucleotides. We present here the 2.6-Å crystal structure of the feline coronavirus (FCoV) Nsp7:Nsp8 complex, which is a 2:1 heterotrimer containing two copies of the ␣-helical Nsp7 with conformational differences between them, and one copy of Nsp8 that consists of an ␣/␤ domain and a long-␣-helix domain. The same stoichiometry is found for the Nsp7:Nsp8 complex in solution, as demonstrated by chemical crosslinking, size exclusion chromatography, and small-angle X-ray scattering. Furthermore, we show that FCoV Nsp8, like its SARSCoV counterpart, is able to synthesize short oligoribonucleotides of up to 6 nucleotides in length when carrying an N-terminal His 6 tag. Remarkably, the same protein harboring the sequence GPLG instead of the His 6 tag at its N terminus exhibits a substantially increased, primer-independent RNA polymerase activity. Upon addition of Nsp7, the RNA polymerase activity is further enhanced so that RNA up to template length (67 nucleotides) can be synthesized. Further, we show that the unprocessed intermediate polyprotein Nsp7-10 of human coronavirus (HCoV) 229E is also capable of synthesizing oligoribonucleotides up to a chain length of six. These results indicate that in case of FCoV as well as of HCoV 229E, the formation of a hexadecameric Nsp7:Nsp8 complex is not necessary for RNA polymerase activity. Further, the FCoV Nsp7:Nsp8 complex functions as a noncanonical RNA polymerase capable of synthesizing RNA of up to template length.

Background: Nucleoprotein (NP) is an essential component of the virus replication complex. Results:The crystal and quaternary structures of Lassa virus NP were determined. Conclusion: Lassa virus NP assembles into a symmetric trimer in solution.Significance: The trimeric complex may have a biological function in the virus life cycle, and its assembly could be a target for antivirals.

Insulin degrading enzyme (IDE) plays key roles in degrading peptides vital in type two diabetes, Alzheimer's, inflammation, and other human diseases. However, the process through which IDE recognizes peptides that tend to form amyloid fibrils remained unsolved. We used cryoEM to understand both the apo- and insulin-bound dimeric IDE states, revealing that IDE displays a large opening between the homologous ~55 kDa N- and C-terminal halves to allow selective substrate capture based on size and charge complementarity. We also used cryoEM, X-ray crystallography, SAXS, and HDX-MS to elucidate the molecular basis of how amyloidogenic peptides stabilize the disordered IDE catalytic cleft, thereby inducing selective degradation by substrate-assisted catalysis. Furthermore, our insulin-bound IDE structures explain how IDE processively degrades insulin by stochastically cutting either chain without breaking disulfide bonds. Together, our studies provide a mechanism for how IDE selectively degrades amyloidogenic peptides and offers structural insights for developing IDE-based therapies.

NAP-1 fold histone chaperones play an important role in escorting histones to and from sites of nucleosome assembly and disassembly. The two NAP-1 fold histone chaperones in budding yeast, Vps75 and Nap1, have previously been crystalized in a characteristic homodimeric conformation. In this study, a combination of small angle X-ray scattering, multi angle light scattering and pulsed electron–electron double resonance approaches were used to show that both Vps75 and Nap1 adopt ring-shaped tetrameric conformations in solution. This suggests that the formation of homotetramers is a common feature of NAP-1 fold histone chaperones. The tetramerisation of NAP-1 fold histone chaperones may act to shield acidic surfaces in the absence of histone cargo thus providing a ‘self-chaperoning’ type mechanism.

Synchrotron radiation circular dichroism, Fourier transform infrared, and nuclear magnetic resonance spectroscopies, and small-angle x-ray scattering were used to monitor the reversible thermal unfolding of hen egg white lysozyme. The results were compared with crystal structures and high- and low-temperature structures derived from molecular-dynamics calculations. The results of both experimental and computational methods indicate that the unfolding process starts with the loss of β-structures followed by the reversible loss of helix content from ∼40% at 20°C to 27% at 70°C and ∼20% at 77°C, beyond which unfolding becomes irreversible. Concomitantly there is a reversible increase in the radius of gyration of the protein from 15 Å to 18 Å. The reversible decrease in forward x-ray scattering demonstrates a lack of aggregation upon unfolding, suggesting the change is due to a larger dilation of hydration water than of bulk water. Molecular-dynamics simulations suggest a similar sequence of events and are in good agreement with the (1)H(N) chemical shift differences in nuclear magnetic resonance. This study demonstrates the power of complementary methods for elucidating unfolding/refolding processes and the nature of both the unfolded structure, for which there is no crystallographic data, and the partially unfolded forms of the protein that can lead to fibril formation and disease.

A setup is presented for automated high-throughput measurements of smallangle X-ray scattering (SAXS) from macromolecular solutions on the bendingmagnet beamline X33 of EMBL at the storage ring DORIS-III (DESY, Hamburg). A new multi-cell compartment allows for rapid switching between in-vacuum and in-air operation, for digital camera assisted control of cell filling and for colour sample illumination. The beamline is equipped with a Pilatus 1 M-W pixel detector for SAXS and a Pilatus 300 k-W for wide-angle scattering (WAXS), and results from the use of the Pilatus detectors for scattering studies are reported. The setup provides a broad resolution range from 100 to 0.36 nm without the necessity of changing the sample-to-detector distance. A new optimized robotic sample changer is installed, permitting rapid and reliable automated sample loading and cell cleaning with a required sample volume of 40 ml. All the devices are fully integrated into the beamline control software system, ensuring fully automated and user-friendly operation (attended, unattended and remote) with a throughput of up to 15 measurements per hour.

Human lipoxygenases (LOXs) and their metabolites have a great impact on human homeostasis and are of interest for targeted drug design. This goal requires detailed knowledge of their structures and an understanding of structure-function relationship. At the moment, there are two complete crystal structures for mammalian LOX [rabbit 12/15LOX (r-12/15LOX) and human 5LOX (h-5LOX)] and a fragment of human 12LOX. The low-resolution structures in solution for various LOX isoforms have brought about controversial results. Here we explored the behavior of r-12/15LOX in aqueous solution under different conditions (salt and pH) by small-angle X-ray scattering (SAXS) and compared it with human platelet-type 12S-LOX (hp-12LOX) and h-5LOX. Thermodynamic calculations concerning the stability of molecular assemblies, thermal motion analysis [TLSMD (translation, libration, and screw rotation motion detection based on crystallographic temperature factor B j )], and results of SAXS analyses brought about the following conclusions: (i) in contrast to its crystal structure, r-12/15LOX functions as a monomer that dominates in solution; (ii) it dimerizes at higher protein concentrations in the presence of salt and with increasing degree of motional freedom of the N-terminal PLAT domain, as suggested by the Y98,614→ R double mutant; (iii) in aqueous solutions, hp-12LOX is stable as a dimer, in contrast to h-5LOX and r-12/15LOX, which are monomeric; and (iv) all three *Corresponding author. E-mail address: ewa.skrzypczak-jankun@utoledo.edu. Present address: A. M. Aleem, Biochemistry Department, College of Sciences, King Saud University, Riyadh 11451, Kingdom of Saudi Arabia.Abbreviations used: LOX, lipoxygenase; r-12/15LOX, rabbit 12/15LOX; h-5LOX, human 5LOX; SAXS, small-angle X-ray scattering; hp-12LOX, human platelet-type 12S-LOX; PDB, Protein Data Bank. doi:10.1016/j.jmb.2011.04.035 J. Mol. Biol. (2011

A novel integrative bioinspired surface with wettable patterns and gradient (WPGS) is proposed for fog collection via a novel anodic oxidation strategy. We study the water collection behaviors on WPGS with different parameters. Quantitative force analysis is presented, providing evidence for the underlying mechanism leading to the directional motion of the droplet, which is consistent with the experimental results. Such a surface can not only improve the fog droplet capture performance effectively owing to wettable patterns but also accelerate surface regeneration by taking full advantage of the cooperation of multidriving forces, leading to a further fog collection enhancement.