Materials and methodsThe sequences for the different transporters were subcloned into various pET vectors (Novagen), which attached poly---histidine tags at the amino---or carboxy---terminus. The constructs were expressed in E. coli BL21 (DE3) (Stratagene) in a 50---liter fermenter at 37°C and were induced using 1 mM IPTG. BtuC and BtuD were coexpressed from a single plasmid, with the BtuC subunit containing an NH 2 ---terminal decahistidine tag. BtuCD protein was solubilized in 1% dodecyl---N,N---dimethylamineoxide (LDAO) and purified using Ni---NTA metal affinity chromatography (Qiagen), followed by gel filtration. Crystals were grown by mixing BtuCD protein (20 mg/ml) with an equal volume of reservoir solution (100 mM Tris pH 8.0, 300 mM magnesium nitrate, 21% polyethylene glycol 2000, 0.8% 2---methyl---2,4---pentanediol in D 2 O) in sitting drops. Crystals grew to a final size of 0.1 0.15 0.5 mm 3 in a week, and were frozen in liquid N 2 before data collection. Cells expressing selenomethionine BtuCD protein were grown in M9 medium supplemented with seleno---D,L---methionine. Purification and crystallization of the selenomethionine protein was as described above for the native protein. Data sets were collected at the Advanced Photon Source (APS) or at the Stanford Synchrotron Radiation Laboratory (SSRL).

High resolution x-ray diffraction data from crystals of the Rhodobacter sphaeroides photosynthetic reaction center (RC) have been collected at cryogenic temperature in the dark and under illumination, and the structures were refined at 2.2 and 2.6 angstrom resolution, respectively. In the charge-separated D+QAQB- state (where D is the primary electron donor (a bacteriochlorophyll dimer), and QA and QB are the primary and secondary quinone acceptors, respectively), QB- is located approximately 5 angstroms from the QB position in the charge-neutral (DQAQB) state, and has undergone a 180 degrees propeller twist around the isoprene chain. A model based on the difference between the two structures is proposed to explain the observed kinetics of electron transfer from QA-QB to QAQB- and the relative binding affinities of the different ubiquinone species in the QB pocket. In addition, several water channels (putative proton pathways) leading from the QB pocket to the surface of the RC were delineated, one of which leads directly to the membrane surface.

Mechanosensitive ion channels play a critical role in transducing physical stresses at the cell membrane into an electrochemical response. The MscL family of large-conductance mechanosensitive channels is widely distributed among prokaryotes and may participate in the regulation of osmotic pressure changes within the cell. In an effort to better understand the structural basis for the function of these channels, the structure of the MscL homolog from Mycobacterium tuberculosis was determined by x-ray crystallography to 3.5 angstroms resolution. This channel is organized as a homopentamer, with each subunit containing two transmembrane α helices and a third cytoplasmic α helix. From the extracellular side, a water-filled opening approximately 18 angstroms in diameter leads into a pore lined with hydrophilic residues which narrows at the cytoplasmic side to an occluded hydrophobic apex that may act as the channel gate. This structure may serve as a model for other mechanosensitive channels, as well as the broader class of pentameric ligand-gated ion channels exemplified by the nicotinic acetylcholine receptor.

Materials and methodsMoFe protein from aerobically grown cells of Azotobacter vinelandii was purified as described previously (1). Crystals were obtained by equilibrating a reservoir solution containing 13% polyethylene glycol 8000, 1 M sodium chloride and 0.1 M Tris-hydroxyethylaminomethane/HCl buffer at pH 8.0 against 8 µl of a 1:1 mixture of 30 mg/ml of MoFe protein and the reservoir solution under strictly anaerobic conditions. For flash-cooling, the crystals were

Structural data show that the light atom at the center of the nitrogenase active site cofactor is a carbon.

No abstract

The molybdenum-containing enzyme sulfite oxidase catalyzes the conversion of sulfite to sulfate, the terminal step in the oxidative degradation of cysteine and methionine. Deficiency of this enzyme in humans usually leads to major neurological abnormalities and early death. The crystal structure of chicken liver sulfite oxidase at 1.9 A resolution reveals that each monomer of the dimeric enzyme consists of three domains. At the active site, the Mo is penta-coordinated by three sulfur ligands, one oxo group, and one water/hydroxo. A sulfate molecule adjacent to the Mo identifies the substrate binding pocket. Four variants associated with sulfite oxidase deficiency have been identified: two mutations are near the sulfate binding site, while the other mutations occur within the domain mediating dimerization.

Crystal structures of heparin-derived tetra- and hexasaccharides complexed with basic fibroblast growth factor (bFGF) were determined at resolutions of 1.9 and 2.2 angstroms, respectively. The heparin structure may be approximated as a helical polymer with a disaccharide rotation of 174 degrees and a translation of 8.6 angstroms along the helix axis. Both molecules bound similarly to a region of the bFGF surface containing residues asparagine-28, arginine-121, lysine-126, and glutamine-135, the hexasaccharide also interacted with an additional binding site formed by lysine-27, asparagine-102, and lysine-136. No significant conformational change in bFGF occurred upon heparin oligosaccharide binding, which suggests that heparin primarily serves to juxtapose components of the FGF signal transduction pathway.