Karl D. Hardman

Manganese and calcium binding sites of concanavalin A

Abstract The atomic co-ordinates of concanavalin A, a lectin which specifically binds saccharides containing mannosyl and glucosyl residues, have been refined with the fast Fourier least-squares method of Agarwal (1978), with X-ray crystallographic data to 1.75 A. This metalloprotein contains six co-ordinated Mn 2+ and seven co-ordinated Ca 2+ which are 4.25 A apart and are close to the carbohydrate binding site. Both ions are pseudo-octahedral. This is possible for calcium since the average position of two oxygens (from the same carboxyl group) forms one apex of the octahedron. The refinement has improved this region considerably. The ligands for Mn 2+ are five oxygens, three from carboxyl side-chains and two from water molecules, and one nitrogen from a histidyl side-chain. The co-ordination sphere for Ca 2+ contains seven oxygens, three from two carboxyl side-chains, one from a peptide carbonyl, one from an amide side-chain and, as does Mn 2+ , two from water molecules. The averages for the five Mn 2+ O and the seven Ca 2+ O bonds are 2.28 ± 0.04 (standard deviation) and 2.45 ± 0.09, respectively. For Mn 2+ , this average is 0.10 A higher than that found for the Mn(II)hexa-aquoion and is toward the high end of the range usually found for Mn 2+ O complexes. This is of particular significance since Mn 2+ is used as a probe for nuclear magnetic resonance spectroscopic studies of native Mn 2+ - and Mn 2+ -substituted metalloproteins. Also, refinement confirms the non-proline cis peptide bond, which is immediately adjacent to the Ca 2+ and the carbohydrate binding sites. For the final model, the bond lengths and angles for backbone atoms have root-mean-square deviations from standard values of 0.030 A and 5.2 deg., and side-chain atoms have slightly lower values. The crystallographic residual, R factor, for this model is 0.167 for 22,042 structure factors.

Design of a diffractometer and flow cell system for X-ray analysis of crystalline proteins with applications to the crystal chemistry of ribonuclease-S

A flow cell in which a protein crystal is embedded in a changeable liquid medium during X-ray diffraction studies has proved useful in investigating the crystal chemistry of ribonuclease-S. The cell consists of a small polyethylene tube held in a brass yoke which in turn is held by a standard goniometer head. The crystal is prevented from moving by nesting it in cotton linters or particles of Sephadex. Serious scatter of X-rays from the cell and the liquid is reduced to nominal levels by careful arrangement of the collimating system of the diffractometer and by a special counting procedure which gives tolerance of settings without undue increase of noise level. Applications presented include: studies of diffusion rates of solvent and inhibitors into and out of the crystal with time constants ranging from ninety seconds for ammonium sulfate to many hours for iodinated nucleotides; pH effects on unit-cell constants; curves of binding of iodinated inhibitors as a function of external concentration; comparison of binding sites of various inhibitors; interaction of platinum compounds with the enzyme; catalyzed photo-oxidation of specific residues in situ; and measurements of intensity changes due to heavy-atom binding for use in phasing the protein reflections. The flow cell method is particularly useful in studies where changes of intensity rather than absolute values are of prime importance.