Structure of dimeric and monomeric erabutoxin a refined at 1.5 A resolution.

Erabutoxin a has been crystallized in its monomeric and dimeric forms. The structures were refined at 1.50 and 1.49 A resolution, respectively, using synchrotron radiation data. The crystals belong to space group P212121, with cell dimensions a = 49.84, b = 46.62, c = 21.22 A for the monomer and a = 55.32, b = 53.54, c = 40.76 A for the dimer. Using starting models from earlier structure determinations, the monomeric structure refined to an R value of 16.7% (8004 unique reflections, 17.0-1.50 A resolution range), while the dimeric structure has been solved by the molecular-replacement method with a final R value of 16.9% (19 444 unique reflections, 17.4-1.49 A resolution range). The high-resolution electron-density maps clearly revealed significant discrete disorder in the proteins and allowed an accurate determination of the solvent structure. For the monomer, the side chains of six residues were modelled with alternate conformers and 106 sites for water molecules and one site for a sulfate ion were included in the final model, whereas for the dimer, 206 sites for water molecules were included and both C-terminal residues together with the side chains of 11 residues adopted alternative conformations. A comparison was made with earlier structure determinations. The features of the solvent structure of the erabutoxin molecules are discussed in detail.

Crystallization and preliminary X-ray diffraction studies of DNA polymerase from the thermophilic archaeon Sulfolobus solfataricus.

The thermophilic and thermostable family B DNA polymerase from the archaeon Sulfolobus solfataricus (Mr of about 100 kDa) has been crystallized by the hanging-drop vapour-diffusion method at 294 K using ammonium sulfate as precipitant. The crystals belong to the monoclinic space group C2 with cell dimensions a = 187.4, b = 68.5, c = 125.8 A and beta = 107.8 degrees and diffract up to 2.7 A resolution on a rotating-anode X-ray source. Native data have been collected at 100 K. A heavy-atom derivative search is in progress.

Two different crystal forms of sorcin, a penta-EF-hand Ca2+-binding protein.

Sorcin is a 198 amino-acid Ca(2+)-binding protein that belongs to the penta-EF-hand family. Its Ca(2+)-binding domain (residues 33-198) has been crystallized in the absence of Ca(2+) in two different crystal forms. Two complete data sets have been collected on a synchrotron source under cryocooling conditions from crystals grown using ammonium sulfate as precipitant: monoclinic crystals in space group C2, with unit-cell parameters a = 130.93, b = 103.85, c = 78.55 A, beta = 118.0 degrees, diffracting to 2.1 A, and tetragonal crystals in space group P42(1)2, with unit-cell parameters a = b = 103.33, c = 79.15, diffracting to 2.7 A. Crystals were also grown using PEG 6000 as precipitating agent. They also belong to space group C2, diffract to 2.8 A and their unit-cell parameters are very similar to the first form. Structure determination by molecular replacement has been initiated. Structural information should be useful for elucidating the interaction of sorcin with membrane targets.

Crystallization and X-ray diffraction measurements of a thermophilic archaeal recombinant amidase from Sulfolobus solfataricus MT4.

Recombinant amidase is a 55.8 kDa enzyme from the thermophilic archaeon Sulfolobus solfataricus MT4 that catalyses the hydrolysis of aliphatic amides of 2-6 C atoms as well as many aromatic amides. Single crystals of purified amidase were obtained by the hanging-drop method at 294 K. Diffraction data for the native protein (2.55 A resolution) and a putative derivative (2.20 A) have been collected at low temperature using synchrotron radiation. The crystals belong to the rhombohedral space group R3. Structure determination by multiple isomorphous replacement is in progress. It is expected that structural information from this signatured thermostable amidase will increase our knowledge of the molecular mechanisms employed to maintain high-temperature stability in thermophilic proteins.

RAS residues that are distant from the GDP binding site play a critical role in dissociation factor-stimulated release of GDP.

We have previously shown that a conserved glycine at position 82 of the yeast RAS2 protein is involved in the conversion of RAS proteins from the GDP- to the GTP-bound form. We have now investigated the role of glycine 82 and neighbouring amino acids of the distal switch II region in the physiological mechanism of activation of RAS. We have introduced single and double amino acid substitutions at positions 80-83 of the RAS2 gene, and we have investigated the interaction of the corresponding proteins with a yeast GDP dissociation stimulator (SDC25 C-domain). Using purified RAS proteins, we have found that the SDC25-stimulated conversion of RAS from the GDP-bound inactive state to the GTP-bound active state was severely impaired by amino acid substitutions at positions 80-81. However, the rate and the extent of conversion from the GDP- to the GTP-bound form in the absence of dissociation factor was unaffected. The insensitivity of the mutated proteins to the dissociation factor in vitro was paralleled by an inhibitory effect on growth in vivo. The mutations did not significantly affect the interaction of RAS with adenylyl cyclase. These findings point to residues 80-82 as important determinants of the response of RAS to GDP dissociation factors. This suggests a molecular model for the enhancement of nucleotide release from RAS by such factors.