Bibliothek

Notizen: A new X-ray crystallographic beamline is operational at the MAX II synchrotron in Lund. The beamline has been in regular use since August 1998 and is used both for macro- and small molecule diffraction as well as powder diffraction experiments. The radiation source is a 1.8 T multipole wiggler. The beam is focused vertically by a bendable mirror and horizontally by an asymmetrically cut Si(111) monochromator. The wavelength range is 0.8–1.55 Å with a measured flux at 1 Å of more than 1011 photons s−1 in 0.3 mm × 0.3 mm at the sample position. The station is currently equipped with a Mar345 imaging plate, a Bruker Smart 1000 area CCD detector and a Huber imaging-plate Guinier camera. An ADSC 210 area CCD detector is planned to be installed during 2000.

Notizen: Human dUTPase, expressed in Escherichia coli, has been crystallized. Single crystals were obtained by the vapour-diffusion technique using 2-propanol and PEG 4000 as precipitants. The enzyme was co-crystallized with the substrate dUTP and a metal chelator EDTA to prevent hydrolysis of the substrate. The crystals belong to the orthorombic space group P212121 with cell dimensions a = 67.51, b = 68.26 and c = 91.00 Å. The crystallographic asymmetric unit contains one trimer of identical subunits.

Notizen: The Escherichia coli molecular chaperone cpn60 oligomer, [cpn60]14, also called GroEL, has been crystallized and examined by X-ray crystallography and self-rotation function calculations. The crystals show unit-cell dimensions a = 143.3, b = 154.6 and c = 265 Å, with α = 82, β = 95 and γ = 107°. The space group is P1 and crystals diffract to 7 Å. X-ray analysis shows that the oligomer has one sevenfold symmetry axis and seven twofold axes that are all perpendicular to the sevenfold. The symmetry suggests that [cpn60]24 consists of two heptamers, [cpn60]7, stacked on top of each other. The orientations of the symmetry axes of the two independent [cpn60]14 oligomers in the triclinic unit cell have been determined relative to the crystallographic axes. The two oligomers in the unit cell are arranged side-by- side, but the second oligomer is rotated 26° around the sevenfold axis relative to the first oligomer.

Notizen: The X-ray crystal structure of a uridine vanadate-ribonuclease A complex has been determined at 1.3 Å resolution. The resulting structure includes all 124 amino-acid residues, a uridine vanadate, 131 water molecules, and a single bound 2-methyl-2-propanol. Side chains of 11 surface residues showing discrete disorder were modeled with multiple conformations. The final crystallographic R factor is 0.197. Structures obtained from high-level ab initio quantum calculations of model anionic oxyvanadate compounds were used to probe the effects of starting structure on the refinement process and final structure of the penta-coordinate phosphorane analog, uridine vanadate. The least-squares refinement procedure gave rise to the same final structure of the inhibitor despite significantly different starting models. Comparison with the previously determined complex of ribonuclease A with uridine vanadate obtained from a joint X-ray/neutron analysis (6RSA) [Wlodawer, Miller & Sjölin (1983). Proc. Natl Acad. Sci. USA, 80, 3628–3631] reveals similarities in the overall enzyme structure and the relative position of the key active-site residues, Hisl2, His119 and Lys41, but significant differences in the V—O bond distances and angles. The influence of ligand binding on the enzyme structure is assessed by a comparison of the current X-ray structure with the phosphate-free ribonuclease A structure (7RSA) [Wlodawer, Svensson, Sjölin & Gilliland (1988). Biochemistry, 27, 2705–2717]. Ligand binding alters the solvent structure, distribution and number of residues with multiple conformations, and temperature factors of the protein atoms. In fact, the temperature factors of atoms of several residues that interact with the ligand are reduced, but those of the atoms of several residues remote from the active site exhibit marked increases.

Notizen: Three procedures, or `tools', have been developed and tested for applying maximum-entropy methods to phase extension and to ab initio phase determination. The phase expander tool has been used in connection with the solution of two previously unknown macromolecular structures. An efficient algorithm for the determination of an electron-density distribution that is everywhere positive and that agrees with observed structure amplitudes (tools II and III) has been used to determine the phases of X-ray diffraction data from recombinant bovine chymosin, a protein with 323 amino-acid residues in the molecular chain, the structure of which was recently determined using replacement methods. By use of the same maximum-entropy methods, the structure amplitudes from the unknown structure of bovine heart creatine kinase, a protein with 381 amino-acid residues, have been phased ab initio to 2.7 Å resolution. The phases of the centric reflections have also been confirmed by a satisfactory solution of the Patterson map of a mercury derivative. The current status of the structure interpretation is presented. This technique has also been applied to a test case where 48 centric reflections from bovine prothrombin fragment 1 data were phased ab initio and subsequently used in the determination of Patterson solutions for a heavy-atom derivative data set.

Notizen: The cross vectors of the native Patterson map are shown to exhibit non-crystallographic symmetry in the case of local axes parallel to one another. This information can be used to determine the translation component of such axes. A program is described to search for this cross vector, and is tested on low-resolution data from crystals of the tetradecameric GroEL molecule, the decameric GTP cyclohydrolase I and the tetradecameric proteosome. For GroEL, the function produces a packing arrangement optimal for sevenfold symmetry, and is in agreement with the dimensions of the molecule as given by electron microscopy data and the recently determined crystal structure. Positioning of local axes is confirmed by two high-resolution crystal structure analyses: the fivefold axis in cyclohydrolase I and the sevenfold axis in the proteosome. Implications for the location of heavy-atom positions are discussed for these two cases.

Notizen: An efficient algorithm for the determination of an everywhere positive electron-density distribution that agrees with observed structure amplitudes has been used to determine the phases of X-ray diffraction data from recombinant bovine chymosin, a protein with 323 amino-acid residues in the molecular chain whose structure was recently determined using molecular replacement methods. A systematic procedure for testing the signs of centric reflections, using the total entropy of the map as a figure of merit, was used to produce a low-resolution map. The phases of acentric and additional centric reflections were then chosen by adding them to the map with various possible phases and computing the total entropy of the resulting map. Of 159 centric reflections whose phases were chosen by this procedure, 141 had the same phase as in the refined structure. The median absolute phase difference for 1811 acentric reflections was 32°. A map produced from these 1970 reflections, out of 12 346 reflections in the data set, showed a remarkable agreement with the refined structure. This molecule is many times larger than any whose structures have previously been determined without the use of isomorphous replacement, molecular replacement or anomalous dispersion, and the map demonstrates the potential of maximum-entropy methods in macromolecular structure determination.

Notizen: Zusammenfassung Die Identifizierung und der Bildungsmechanismus des fluoreszierenden Produktes aus Glyoxylsäure und Tryptamin wird beschrieben. Eine intramolekulare säure-katalysierte Reaktion wird für die Bildung des fluoreszierenden Produktes vorgeschlagen.

Notizen: Summary N-acetylated and tertiary indolamines, some of which are possible neurotransmitter candidates in the CNS, cannot be visualized with the standard Falck-Hillarp histofluorescence method and very little is known about their cellular localization. The present investigation demonstrates that glyoxylic acid (GA), formaldehyde (FA) in combination with aluminum ions (the ALFA method) and trifluoroacetic acid anhydride (TFAA) are capable of forming fluorescent compounds from N-acetylated (e.g. melatonin and N-acetyl-5-hydroxytryptamine) and tertiary (e.g. bufotenin) indolamines in histochemical protein models. With GA and FA-aluminum more vigorous reaction conditions were required for demonstration of these compounds compared to those needed for optimal visualization of primary catecholand indolamines (prolonged reaction time and higher concentration of GA and FA and aluminum ions). The fluorophore formation from N-acetylated and tertiary indolamines, which represents a new reaction principle in amine fluorescence histochemistry, is proposed to proceed as follows. In the first step, the indole reacts in 2-position with the reagent. The intermediate formed is dehydrated in the second step, yielding a strongly fluorescent 2-methylene derivative, which either per se or as the corresponding autoxidized dimer constitutes the main fluorophore. TFAA and related anhydrides represent new and potent reagents for histochemical visualization of N-acetylated indolamines such as melatonin. In contrast to the GA and ALFA reactions the optimal formation of fluorphores with TFAA required only mild reaction conditions (2–10 min at 0–20° C). The main fluorophore formed from melatonin has been identified and the reaction with TFAA is proposed to proceed as follows. An unstable intermediate, the isoimidinium carboxylate, is formed in the first step and this compound is then cyclized to form the fluorophore, 6-methoxy-1-methyl-3,4-dihydro-β-carboline. The GA and ALFA methods are already widely used for visualization of catecholamine systems. The fluorescence microscopical and microspectrofluorometric analysis did not, however, veveal any specific structures containing N-acetylated or tertiary indolamines in the rat CNS. The TFAA reaction was highly specific for N-acetylated indolamines when applied to protein models. However, in tissue a disturbing background fluorescence appeared, which under all reaction conditions tested, developed concomitantly with the specific fluorescence from melatonin. The problem with this background reaction has to be solved before the TFAA reaction can be applied for demonstration of N-acetylated indolamines in tissue.