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High-throughput crystallography has reached a level of automation where complete computer-assisted robotic crystallization pipelines are capable of cocktail preparation, crystallization plate setup, and inspection and interpretation of results. While mounting of crystal pins, data collection and structure solution are highly automated, crystal harvesting and cryocooling remain formidable challenges towards full automation. To address the final frontier in achieving fully automated high-throughput crystallography, the prototype of an anthropomorphic six-axis universal micromanipulation robot (UMR) has been designed and tested; this UMR is capable of operator-assisted harvesting and cryoquenching of protein crystals as small as 10 microm from a variety of 96-well plates. The UMR is equipped with a versatile tool exchanger providing full operational flexibility. Trypsin crystals harvested and cryoquenched using the UMR have yielded a 1.5 A structure demonstrating the feasibility of robotic protein crystal harvesting.

Research has been conducted on the phosphotriesterase isolated from Pseudomonas diminuta. The high-resolution X-ray structures of the substituted phosphotriesterase forms crystallized in the substrate analogues absence are described.

Research has been conducted on the molecular structure of methylmalonyl CoA decarboxylase from Escherichia coli. The use of the X-ray crystallographic analysis is described.

The molecular structures of the bis(Mg2+)-adenosine triphosphate-oxalate complex of pyruvate kinase were examined. Pyruvate kinase from rabbit muscle was cocrystallized as a complex with MgIIATP, oxalate, magnesium ion and either potassium ion or sodium ion. Results showed that there were negligible differences in the structures of the complexes with potassium ion and sodium ion. It was also found that there is a variety of subunit conformations within the asymmetric unit.

The functional properties of four site-directed vegetative cell ferredoxin (Fd) mutants, C41S, C46S, C49S and C79S, and an X-ray crystallographic structure for C49S are presented. Each mutant has the ability to rapidly accept an electron from deazariboflavin semiquinone and to transfer an electron from its reduced form to oxidized ferredoxin:NADP+ reductase (FNR) although they are less reactive toward FNR and are less stable in solution than is wt Fd. Results suggest that the reactivity of the mutants is not correlated with altered reduction potentials.

Nonconservative mutations of the critical residues S47, F65, and E94, on the surface of ferredoxin have large parallel effects on the reduction potential and the electron-transfer reactivity of the (2Fe-2S) cluster. The reduction potential changes are not the principal factor governing electron transfer reactivity. The specific orientation of the proteins within the transient electron-transfer complex control the kinetic properties Anabaena Ferredoxin.

The three-dimensional structure of 4-chlorobenzoyl-CoA dehalogenase from Pseudomonas sp. strain CBS-3 is studied. Crystallographic analysis showed that the enzyme is a trimer and each subunit folds into two distinct motifs. The bigger, N-terminal domain has 10 strands of beta-pleated sheet that form two distinct layers which are almost perpendicular to each other. Alpha-helices are found on the sides of the layers of beta-sheet. The C-terminal domain is made of three amphiphilic alpha-helices. The two domains of the subunit are linked by a cation.

The phosphotriesterase from Pseudomonas diminuta requires a binuclear metal center for its activity. The enzyme contains two equivalents of zinc per subunit. Three-dimensional Zn2+/Zn2+ substituted structure of the enzyme indicates that the inhibitor diethyl 4-methylbenzylphosphonate binds to a site near the binuclear metal center. The activation of the nucleophilic water molecule which attacks the electrophilic phosphorus of the substrate occurs through interaction with the binuclear metal center.

An X-ray crystallographic analysis of the structure of kanamycin nucleotidyltransferase (KNTase) complexed with AMPCPP and kanamycin reveals that KNTase exists in a dimeric form. The enzyme exhibits both protein-antibiotic and protein-nucleotide interactions. The 4'-hydroxyl group of kanamycin which is appropriately aligned for a displacement attack at the phosphorus of the nucleotide is 5 angstrom apart. Glu at position 145 which is at the active site of the enzyme initially reacts with the kanamycin to activate the antibiotic for further attack at the phosphorus.

X-ray crystallographic studies of the cadmium substituted phosphotriesterase reveal the presence of ions, one ligated by two nitrogen and four oxygens forming a distorted octahedron and the other a trigonal bipyramid with two nitrogen- and three oxygen- containing ligands. Crystallization of the natural protein which has two zinc ions has not been possible. The enzyme has the rare capacity to hydrolyze common pesticides as well as the phosphofluoridates, though the exact mechanism is unknown, and it is an important weapon against chemical warfare.

Single crystal X-ray diffraction studies provide the three-dimensional structure at 2.1 angstroms resolution of phosphotriesterase, an organophosphate-degrading enzyme. The enzyme molecules contain an alpha/beta barrel with eight strands of parallel beta-pleated sheet and two antiparallel beta-strands at the C-terminus. This enzyme can mediate detoxification of organophosphate nerve agents.

Molecular replacement procedures were used to determine the three-dimensional structure of the high-potential iron-sulfur protein (iso-form II), isolated from Ectothiorhodospira vacuolata. The molecules revealed reverse turns that enveloped the iron-sulfur cluster with the 34, 37, 51 and 65 cysteine residues, binding the metal center to the polypeptide chain. Fifty-seven solvent molecules were observed on the protein surface.

Molecular composition of kanamycin nucleotidyltransferase, initially separated from Staphylococcus aureus, was examined using X-ray crystallographic analysis to a resolution 3.0 angstroms. The mixed beta-pleated sheet composed of five strands was the main feature of the N-terminal area, while five alpha-helices were present in the C-terminal region. An ellipsoid with a deep cleft was produced when the two subunits combine together.

The crystal structures of the ligand binding domain (LBD) of the rat vitamin D receptor (VDR) in ternary complexes with a synthetic LXXLL-containing peptide and four ligands are determined. The differences in the biological activity of the 2-carbon-modified analogues are not reflected in the three-dimensional structure of the LBD of the VDR.