Insolubility of full-length HIV-1 integrase (IN) limited previous structure analyses to individual domains. By introducing five point mutations, we engineered a more soluble IN that allowed us generate multidomain crystals. The first is reported. It incorporates the catalytic core and C-terminal domains (residues 52–288). resolved 2.8 Å Y-shaped dimer. Within dimer, form only dimer interface, are located 55 apart. A 26-aa α-helix, α6, links domain core. kink in one two α6 helices occurs near...

As the first structural elucidation of a modular polyketide synthase (PKS) domain, crystal structure macrocycle-forming thioesterase (TE) domain from 6-deoxyerythronolide B (DEBS) was solved by combination multiple isomorphous replacement and multiwavelength anomalous dispersion refined to an R factor 24.1% 2.8-Å resolution. Its overall tertiary architecture belongs α/β-hydrolase family, with two unusual features unprecedented in this family: hydrophobic leucine-rich dimer interface...

The structure of pancreatic cholesterol esterase, an enzyme that hydrolyzes a wide variety dietary lipids, mediates the absorption esters, and is dependent on bile salts for optimal activity, determined to 1.6 Å resolution. A full-length construct, mutated eliminate two N-linked glycosylation sites (N187Q/N361Q), was expressed in HEK 293 cells. Enzymatic activity assays show purified, recombinant, mutant has identical native, glycosylated purified from bovine pancreas. monomeric exhibits...

A wide range of organophosphorus nerve agents, including Soman, Sarin, and Tabun is efficiently hydrolyzed by the phosphotriesterase enzyme diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris. To date, lack available inhibitors DFPase has limited studies on its mechanism. The de novo design, synthesis, characterization substrate analogues acting as competitive are reported. 1.73 Å crystal structure O,O-dicyclopentylphosphoroamidate (DcPPA) bound to shows a direct coordination...

Hydrogen atoms constitute about half of all in proteins and play a critical role enzyme mechanisms macromolecular solvent structure. atom positions can readily be determined by neutron diffraction, as such, diffraction is an invaluable tool for elucidating molecular mechanisms. Joint refinement X-ray data lead to improved models compared with the use alone has now been incorporated into modern, maximum-likelihood based crystallographic programs like CNS. applied collected on crystals...

The 1.1 Å, ultrahigh resolution neutron structure of hydrogen/deuterium (H/D) exchanged crambin is reported. Two hundred ninety-nine out 315, or 94.9%, the hydrogen atom positions in protein have been experimentally derived and resolved through nuclear density maps. A number unconventional interactions are clearly defined, including a potential O─H…π interaction between water molecule aromatic ring residue Y44, as well C─H…O bonds. Hydrogen bonding networks that ambiguous 0.85 Å X-ray can be...

A 1.1 Å resolution, room-temperature X-ray structure and a 2.1 resolution neutron of chitin-degrading lytic polysaccharide monooxygenase domain from the bacterium Jonesia denitrificans (JdLPMO10A) show putative dioxygen species equatorially bound to active site copper. Both structures an elongated density for dioxygen, most consistent with Cu(II)-bound peroxide. The coordination environment is Cu(II). In structures, difference maps reveal N-terminal amino group, involved in copper...

Diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris is an efficient and robust biocatalyst for the hydrolysis of a range highly toxic organophosphorus compounds including nerve agents sarin, soman, cyclosarin. In contrast to substrate diisopropyl fluorophosphate (DFP) possess asymmetric phosphorus atom, which leads pairs enantiomers that display markedly different toxicities. Wild-type DFPase prefers less stereoisomers substrates slower detoxification despite rapid hydrolysis. Enzyme...

Gene silencing mediated by RNA interference requires the sequence-specific recognition of target mRNA endonuclease Argonaute, primary enzymatic component RNA-induced complex. We report crystal structure Aquifex aeolicus refined at 3.2A resolution. Relative to recent Argonaute structures, a 24 degrees reorientation PAZ domain in our opens basic cleft between N-terminal and domains, exposing guide strand binding pocket PAZ. This rearrangement leads branched, Y-shaped system grooves that...

Ultrahigh-resolution structures provide unprecedented details about protein dynamics, hydrogen bonding and solvent networks. The reported 0.70 Å, room-temperature crystal structure of crambin is the highest-resolution ambient-temperature a achieved to date. Sufficient data were collected enable unrestrained refinement associated networks using SHELXL . Dynamic resulting from alternative side-chain conformations shifts in water positions are revealed, demonstrating that polypeptide...

Protein B2 from Nodamura virus (NMV B2), a member of the Nodavirus family, acts as suppressor RNA interference (RNAi). The N-terminal domain NMV B2, consisting residues 1-79, recognizes double-stranded (dsRNA). 2.5 A crystal structure RNA-binding shows dimeric, helical bundle structure. conserved set compared with flock house despite limited sequence identity. packing places along one face symmetry-related molecules, suggesting potential platform for recognition dsRNA.

New developments in macromolecular neutron crystallography have led to an increasing number of structures published over the last decade. Hydrogen atoms, normally invisible most X-ray crystal structures, become visible with neutrons. Using X-rays allows one see structure, while neutrons allow reveal chemistry inherent these structures. A surprising and sometimes controversial results emerged; because it is difficult or predict hydrogen atoms when they are seen by can be unexpected locations...

Viral suppressors of RNA interference (RNAi) appear to have evolved as a response this innate genomic defense. We report the nucleic acid binding properties Cucumovirus RNAi suppressor tomato aspermy virus protein 2B (TAV 2B). Using total internal reflection fluorescence spectroscopy (TIRFS), we show that TAV binds double-stranded corresponding siRNAs and miRNAs, well single-stranded oligonucleotides. A number positively charged residues between amino acids 20 30 are critical for binding....

Diisopropyl fluorophosphatase (DFPase) is a calcium-dependent phosphotriesterase that acts on variety of highly toxic organophosphorus compounds act as inhibitors acetylcholinesterase. The mechanism DFPase has been probed using methods, including isotopic labelling, which demonstrated the presence phosphoenzyme intermediate in reaction mechanism. In order to further elucidate and ascertain protonation states residues solvent molecules active site, neutron structure was solved at 2.2 Å...

The signal-to-noise ratio is one of the limiting factors in neutron macromolecular crystallography. Protein perdeuteration, which replaces all H atoms with deuterium, a method improving crystallography experiments by reducing incoherent scattering hydrogen isotope. Detailed analyses perdeuterated and hydrogenated structures are necessary order to evaluate utility crystals for diffraction studies. room-temperature X-ray structure diisopropyl fluorophosphatase (DFPase) reported at 2.1 Å...

The enzyme diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris is capable of decontaminating a wide variety toxic organophosphorus nerve agents. DFPase structurally related to number enzymes, such as the medically important paraoxonase (PON). In order investigate reaction mechanism this phosphotriesterase and elucidate protonation state active-site residues, large-sized crystals have been prepared for neutron diffraction studies. Available H atoms exchanged through vapour diffusion...

The room-temperature (RT) X-ray structure of H/D-exchanged crambin is reported at 0.85 Å resolution. As one the very few proteins refined with anisotropic atomic displacement parameters two temperatures, dynamics atoms in RT and 100 K structures are compared. Neutron diffraction data from an crystal collected Protein Crystallography Station (PCS) showed beyond 1.1 This highest resolution neutron to date for a protein will reveal important details motions H D structures.

A practical method for operating existing undulator synchrotron beamlines at photon energies considerably higher than their standard range is described and applied beamline 19-ID of the Structural Biology Center Advanced Photon Source enabling operation 30 keV. Adjustments to spectrum were critical enhance keV flux while reducing lower- higher-energy harmonic contamination. Pd-coated mirror Al attenuators acted as effective low- high-bandpass filters. The resulting keV, although...

Three high-resolution X-ray crystal structures of malate dehydrogenase (MDH; EC 1.1.1.37) from the methylotroph Methylobacterium extorquens AM1 are presented. By comparing apo MDH, a binary complex MDH and NAD + , ternary oxaloacetate with ADP-ribose occupying pyridine nucleotide-binding site, conformational changes associated formation catalytic were characterized. While substrate-binding site is accessible in enzyme resting state or -bound forms, substrate-bound form exhibits closed...

Bacteria and fungi express lytic polysaccharide monooxgyenase (LPMO) enzymes that act in conjunction with canonical hydrolytic sugar-processing to rapidly convert polysaccharides such as chitin, cellulose starch single monosaccharide products. In order gain a better understanding of the structure oxidative mechanism these enzymes, large crystals (1-3 mm(3)) chitin-processing LPMO from Gram-positive soil bacterium Jonesia denitrificans were grown screened for their ability diffract neutrons....